The present invention provides novel radiopharmaceuticals useful for the diagnosis of infection and inflammation, reagents and kits useful for preparing the radiopharmaceuticals, methods of imaging sites of infection and/or inflammation in a patient, and methods of diagnosing diseases associated with infection or inflammation in patients in need of such diagnosis. The radiopharmaceuticals bind in vivo to the leukotriene B4 (LTB4) receptor on the surface of leukocytes which accumulate at the site of infection and inflammation. The reagents provided by this invention are also useful for the treatment of diseases associated with infection and inflammation.
The rapid diagnosis of diseases associated with focal infection and inflammation is a currently unmet clinical need. Inflammation is the result of the detection of an abnormality in the body, such as infection, by leukocytes. Leukocytes become activated and gravitate toward the site of the abnormality. When the leukocytes become fully activated they degranulate and release proteolytic enzymes as well as chemoattractants resulting in a chemotactic gradient and as a consequence the recruitment of additional leukocytes. The result is a concentration of activated leukocytes at the site. This localization provides a means for diagnosing diseases associated with infection and inflammation through the use of leukocytes labeled with an externally detectable radioisotope and gamma scintigraphy.
Two approaches have been taken to utilize this mechanism for imaging infection and inflammation. The first involves isolating leukocytes from a patient, labeling the leukocytes with a radioisotope and then reinjecting the radiolabeled autologous leukocytes into the patient. This approach has several drawbacks including the effect of the labeling methodology on the biological activity of the leukocytes manifest as a diminished number of competent leukocytes, and the hazards and inconvenience of handling the patient""s blood. The second approach involves injecting into the patient a radiopharmaceutical that binds to activated leukocytes in vivo.
An example of the in vivo labeling approach is the use of radiolabeled monoclonal antibodies or fragments thereof that are directed against a leukocyte activation marker, as described in Morgan, Jr., U.S. Pat. No. 5,376,356. A leukocyte activation marker is an antigen on the surface of the leukocyte that is poorly expressed or not expressed at all until activation of the leukocyte. This approach suffers from the disadvantages associated with the use of many proteinaceous radiopharmaceuticals as diagnostics, namely, generally slow blood clearance which results in high background activity unless an inconveniently long period of time is allowed to pass between injection and imaging, and the possibility of an allergic reaction by the patient to a foreign protein.
It has been proposed that these problems can be overcome by using radiolabeled peptides that bind in vivo to surface receptors on activated leukocytes (Fischman et. al., Semin. Nucl. Med., 1994, 24, pp 154-168). The chemotactic peptide, fMLF, labeled with In-111 or Tc-99m have been shown to accumulate at sites of infection in experimental animal models. However, the peptide fMLF is a potent agonist for the leukocytes and thus has limited clinical applicability in a diagnostic radiopharmaceutical. The limitations include the potential for serious deleterious effects to the patient, such as a severe drop in white blood cell count, resulting from the activation of the leukocytes upon injection of even small amounts of the potent agonist peptide.
Another alternative approach has been described by Rubin et. al. in U.S. Pat. No. 4,926,869 involving the use of a radiolabeled immunoglobulin or fragment thereof. The immunoglobulin accumulates at the site of infection or inflammation by a non-specific mechanism attributed to the leakage of labeled immunoglobulin from the circulation into the greatly expanded protein space at the site. However, this approach suffers from the same disadvantages associated with the use of a proteinaceous substance as described above.
Therefore, there remains a need for new radiopharmaceuticals for imaging infection and inflammation that have improved pharmacokinetics, especially faster blood clearance, and do not cause serious side-effects in patients.
Leukotriene B4 (LTB4) is synthesized from arachidonic acid by the action of 5-lipoxygenase and leukotriene A4 hydrolase. LTB4 is released by polymorphonuclear leukocytes (PMN), macrophages, mast cells, basophils and monocytes with each cell type having an LTB4 surface receptor. Endothelial cells, eosinophils and platelets do not generate LTB4. The binding of LTB4 to its surface receptor promotes chemotaxis in PMN""s, macrophages and eosinophils. It also induces PMN aggregation, adherence of PMNs to vascular endothelium and PMN diapedesis.
LTB4 in conjunction with PMN, macrophages, mast cells, basophils and monocytes has been implicated in a variety of diseases which involve undesirable inflammatory responses in diverse tissues, including infection, tissue injury and transient ischemia. In the case of reperfusion injury and transplant rejection, LTB4 together with PMN, macrophages and mast cells have been causally demonstrated to play a major role in the inflammatory processes associated with these phenomena. In addition, LTB4 in conjunction with PMN, macrophages, mast cells, basophils plays a pivotal role in the development of inflammatory bowel disease. Colonic mucosal scrapings from inflammatory bowel disease patients generate 6 fold more LTB4 than from corresponding normal subjects. Thus a radiopharmaceutical which binds to the LTB4 receptor at sub-therapeutic levels should be able to rapidly detect inflammatory disease processes throughout the body.
In the present invention it has been found that radiopharmaceuticals capable of binding to the LTB4 receptor are useful for imaging sites of infection and inflammation.
The present invention provides novel radiopharmaceuticals useful for the diagnosis of infection and inflammation, reagents and kits useful for preparing the radiopharmaceuticals, methods of imaging sites of infection and/or inflammation in a patient, and methods of diagnosing diseases associated with infection or inflammation in patients in need of such diagnosis. The radiopharmaceuticals bind in vivo to the leukotriene B4 (LTB4) receptor on the surface of leukocytes which accumulate at the site of infection and inflammation. The reagents of this invention are also useful in the treatment of diseases associated with infection and inflammation.
The radiopharmaceuticals of the present invention are small molecules and so do not suffer from the disadvantages associated with radiolabeled proteins or antibodies. As antagonists, the radiopharmaceuticals have significantly diminished risk of producing side-effects. The radiopharmaceuticals of the present invention have utility in the rapid detection of inflammatory or infectious diseases such as inflammatory bowel, fever of unknown origin, reperfusion injury and transplant rejection. The reagents of this invention are useful in the treatment of diseases associated with infection and inflammation.
The present invention also provides for novel dual isotope imaging methods utilizing LTB4-targeted imaging reagents in combination with perfusion imaging radiopharmaceuticals, such as cardiac or brain perfusion agents. The combination of imaging agents in the simultaneous dual isotope imaging method of this invention is useful for the concurrent imaging of organ blood flow and sites of inflammation which may be associated with disease processes such as reperfusion injury, atherosclerosis or infection in the organ of interest.
[1] In an embodiment, the present invention provides a method of concurrent imaging in a mammal comprising:
a) administering to said mammal a radiolabeled LTB4 binding agent and a radiolabeled perfusion imaging agent; and
b) concurrently detecting the radiolabeled LTB4 binding agent bound at the LTB4 receptor and the radiolabeled perfusion imaging agent;
wherein said radiolabeled agents have spectrally separable energies.
[2] In an embodiment, the present invention provides a method according to embodiment [1] for use in concurrent imaging sites of inflammation and organ perfusion.
[3] In an embodiment, the present invention provides a method according to embodiment [1] for use in diagnosing and localizing sites of inflammation and perfusion abnormalities.
[4] In an embodiment, the present invention provides a method according to embodiment [1] for use in concurrent detection and localization of sites of ischemic tissue injury and perfusion abnormalities.
[5] In an embodiment, the present invention provides a method according to embodiment [1] for use in concurrent detection and localization of sites of ischemic tissue injury (e.g. reperfusion injury), vulnerable plaque, bacterial endocarditis or cardiac transplant rejection.
[6] In an embodiment, the present invention provides a method according to embodiment [1] for use in the concurrent detection and localization of sites of vulnerable plaque and perfusion abnormalities.
[7] In an embodiment, the present invention provides a method according to embodiment [1] for use in the concurrent detection and localization of sites of cardiac infection and perfusion abnormalities.
[8] In an embodiment, the present invention provides a method according to embodiment [1] for use in the concurrent detection and localization of sites of cardiac transplant rejection and perfusion abnormalities.
[9] In an embodiment, the present invention provides a method according to embodiment [1] wherein the method of detecting the radiolabeled agents is by scintigraphic imaging.
[10] In an embodiment, the present invention provides a method according to embodiment [1] wherein the method of detecting the radiolabeled agents is by radiation detecting probe.
[11] In an embodiment, the present invention provides a method according to embodiment [1] wherein the method of detecting the radiolabeled agents is by planar or ring gamma camera.
[12] In an embodiment, the present invention provides a method according to embodiment [1] wherein administering said radiolabeled LTB4 binding agent and radiolabeled perfusion imaging agent is concurrent.
[13] In an embodiment, the present invention provides a method according to embodiment [1] wherein administering said radiolabeled LTB4 binding agent and radiolabeled perfusion imaging agent is sequential.
[14] In an embodiment, the present invention provides a method according to embodiment [1] wherein the radiolabeled LTB4 binding agent is administered in a synergystically effective amount.
[15] In an embodiment, the present invention provides a method according to embodiment [1] wherein detecting the radiolabeled LTB4 binding agent and the radiolabeled perfusion imaging agent is enhanced to afford improved accuracy of the image registration.
[16] In an embodiment, the present invention provides a method according to embodiment [1] wherein the energies of the radiolabeled agents are spectrally separable by pulse-height analysis.
[17] In an embodiment, the present invention provides a method according to embodiment [1] wherein the difference in spectral energies of the radiolabeled agents is  greater than 10 Kev.
[18] In an embodiment, the present invention provides a method of any one of embodiments [1-17] wherein the radiolabeled LTB4 binding agent is radiolabeled with a radioisotope selected from the group consisting of 99mTc, 111In, 95Tc, 62Cu, 67Ga, 68Ga, 123I, 125I, 18F, 11C, 13N, 15O, and 75Br.
[19] In an embodiment, the present invention provides a method of any one of embodiments [1-17] wherein the LTB4 binding agent is radiolabeled with In-111 Tc-99m or I-123.
[20] In an embodiment, the present invention provides a method of any one of embodiments [1-17] wherein the perfusion imaging agent is radiolabeled with Tc-99m or Tl-201.
[21] In an embodiment, the present invention provides a method of any one of embodiments [1-17] wherein the perfusion imaging agent is hexakis methoxyisobutyl isonitrile Technetium(I) (99mTc-Sestamibi), 210Tl, 99mTc-tetrofosmin, 99mTc-furifosmin, or 99mTc-NOET.
[22] In an embodiment, the present invention provides a method of any one of embodiments [1-17] wherein the radiolabeled LTB4 binding agent is a reagent capable of direct transformation into a compound radiolabeled with a radioisotope selected from the group consisting of 99mTc, 111In, 95Tc, 62Cu, 67Ga, and 68Ga and said reagent having the formula:
Wexe2x80x94Xxe2x80x94Lnxe2x80x94Yxe2x80x94Lnxe2x80x2xe2x80x94Ch, Wexe2x80x94Xxe2x80x94Ln(Lnxe2x80x2xe2x80x94Ch)xe2x80x94Y, or Zxe2x80x94Lnxe2x80x2xe2x80x94Ch, 
wherein,
We is selected from the group: 
xe2x80x83wherein,
A1 is N, Cxe2x80x94OH, or CH;
A2 and A3 are independently N or CH;
A4 is N or CR3;
A5 is O or S;
A6 is O, CH2 or S;
A7 is Cxe2x80x94OH, N, NH, O or S;
A8 is NH, CH2, O, S, N, or CH;
A9 is N or CH;
a and b indicate the alternative positions of a double bond;
R1 is selected from the group: H, xe2x80x94C(xe2x95x90NH)NH2, C1-C6 alkyl substituted with 0-3 R4, C1-C6 alkoxy substituted with 0-3 R4, aryl substituted with 0-3 R5, and heterocycle substituted with 0-3 R5;
R2 is selected from the group: H, C1-C3 alkyl, C2-C3 alkenyl, cyclopropyl, cyclopropylmethyl, and aryl substituted with 0-3 R5;
R3 is xe2x80x94H, xe2x80x94OH or C1-C3 alkoxy;
or alternatively, R1 and R3 can be taken together with the atoms to which they are attached to form a fused phenyl ring substituted with 0-3 R5;
R4 is independently selected from the group: xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x95x90O, xe2x80x94N(R6)(R7), and xe2x80x94CF3;
R5 is independently selected from the group: xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94N(R6)(R7), xe2x80x94CF3, C1-C3 alkyl, C1-C3 alkoxy, and methylenedioxy;
R6 and R7 are independently H or C1-C3 alkyl; provided that when A1 and A2 are CH, A3 is Cxe2x80x94X, and A4 is CR3, R1 is selected from the group: C1-C5 alkyl substituted with 1-3 R4, C1-C5 alkoxy substituted with 0-3 R4, and aryl substituted with 0-3 R5;
X is O, S, CH2 or CHxe2x95x90CH;
Ln is a linking group having the formula
(CR8R9)gxe2x80x94(W1)hxe2x80x94(M1)kxe2x80x94(CR10R11)g, 
xe2x80x83wherein,
R8, R9, R10 and R11 are independently selected at each occurrence from the group: a bond to Lnxe2x80x2, H, C1-C5 alkyl, and C1-C5 alkoxy, or alternatively, R8 and R9 or R10 and R11 may be taken together to form a 3-6 membered cycloalkyl or heterocycle;
W1 is independently selected from the group: O, S, C(xe2x95x90O)O, OC(xe2x95x90O), CHxe2x95x90CH, (OCH2CH2)p and (CH2CH2O)pxe2x80x2, wherein p and pxe2x80x2 are independently 1-3;
M1 is selected from the group: phenyl substituted with 0-3 R12 heterocycle substituted with 0-3 R12, benzophenone substituted with 0-3 R12, and diphenylether substituted with 0-3 R12;
R12 is independently selected from the group: a bond to Lnxe2x80x2, xe2x80x94COOR13, C1-C5 alkyl substituted with 0-3 R14, and C1-C5 alkoxy substituted with 0-3 R14;
R13 is H or C1-C5 alkyl;
R14 is independently selected from the group: a bond to Lnxe2x80x2, and xe2x80x94COOH;
g is 0-10;
h is 0-3;
k is 0-1;
gxe2x80x2 is 0-5;
provided that when h is 0 and k is 0, g is  greater than 1;
and provided that when W1 is O or S and k is 0, g+gxe2x80x2 is  greater than 1;
Y is selected from C(xe2x95x90O)NH, NHC(xe2x95x90O), Cxe2x95x90O, C(xe2x95x90O)O, OC(xe2x95x90O), NHS(xe2x95x90O)2, C(xe2x95x90O)NHS(xe2x95x90O)2, COOH, C(xe2x95x90O)NH2, NH(Cxe2x95x90O)NH, or tetrazole;
provided that from 0-1 of R9, R10, R11, R12, and R14 is a bond to Lnxe2x80x2 and when one of these variables is a bond to Lnxe2x80x2, then Y is COOH, C(xe2x95x90O)NH2, or tetrazole;
Lnxe2x80x2 is a linking group having the formula:
(W2)hxe2x80x2xe2x80x94(CR15R16)gxe2x80x3xe2x80x94(M2)kxe2x80x2xe2x80x94(W2)hxe2x80x3xe2x80x94(CR17R18)gxe2x80x3xe2x80x2xe2x80x94(W2)hxe2x80x3xe2x80x2
xe2x80x83wherein,
W2 is independently selected at each occurrence from the group: O, S, NH, NHC(xe2x95x90O), NHC(xe2x95x90O)M2, C(xe2x95x90O)NH, C(xe2x95x90O), C(xe2x95x90O)O, OC(xe2x95x90O), NHC(xe2x95x90O)NH, SO2, (OCH2CH2)s, (CH2CH2O)sxe2x80x2, (OCH2CH2CH2)sxe2x80x3, (CH2CH2CH2O)t, and (aa)txe2x80x2, wherein aa is independently at each occurrence an amino acid, and s, sxe2x80x2, sxe2x80x3, t, and xe2x80x2 are independently 1-10;
M2 is selected from the group: aryl substituted with 0-3 R19, cycloalkyl substituted with 0-3 R19, and heterocycle substituted with 0-3 R19;
R15, R16, R17 and R18 are independently selected at each occurrence from the group: xe2x95x90O, COOH, SO3H, PO3H, C1-C5 alkyl substituted with 0-3 R19, aryl substituted with 0-3 R19, benzyl substituted with 0-3 R19, and C1-C5 alkoxy substituted with 0-3 R19, NHC(xe2x95x90O)R20, C(xe2x95x90O)NHR20, NHC(xe2x95x90O)NHR20, NHR20, R20, and a bond to Ch;
R19 is independently selected at each occurrence from the group: COOR20, OH, NHR20, SO3H, PO3H, aryl substituted with 0-3 R20, heterocycle substituted with 0-3 R20, C1-C5 alkyl substituted with 0-1 R21, C1-C5 alkoxy substituted with 0-1 R21 and a bond to Ch;
R20 is independently selected at each occurrence from the group: H, aryl substituted with 0-1 R21 heterocycle substituted with 0-1 R21 cycloalkyl substituted with 0-1
R21 polyalkylene glycol substituted with 0-1 R21 carbohydrate substituted with 0-1 R21 cyclodextrin substituted with 0-1 R21 amino acid substituted with 0-1
R21 polycarboxyalkyl substituted with 0-1 R21, polyazaalkyl substituted with 0-1 R21, peptide substituted with 0-1 R21, wherein said peptide is comprised of 2-10 amino acids, and a bond to Ch;
R21 is a bond to Ch;
kxe2x80x2 is 0-2;
hxe2x80x2 is 0-2;
hxe2x80x3 is 0-5;
hxe2x80x3xe2x80x2 is 0-2;
gxe2x80x3 is 0-10;
gxe2x80x3xe2x80x2 is 0-10;
Ch is a metal bonding unit having a formula selected from the group: 
xe2x80x83wherein:
Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are independently selected at each occurrence from the group: NR22, NR22R23, S, SH, O, OH, PR22, PR22R23, P(NR24)R25R26, P(O)R25R26, and P(S)R25R26;
E is a bond, CH, or a spacer group selected from the group: C1-C10 alkyl substituted with 0-3 R27, aryl substituted with 0-3 R27, cycloalkyl substituted with 0-3 R27, heterocycloalkyl substituted with 0-3 R27, aralkyl substituted with 0-3 R27, and alkaryl substituted with 0-3 R27;
Ea is a C1-C10 alkyl group or a C3-C14 carbocycle; R22, R23, and R24 are each independently selected from the group: a bond to Lnxe2x80x2, hydrogen, C1-C10 alkyl substituted with 0-3 R27, aryl substituted with 0-3 R27, cycloalkyl substituted with 0-3 R27, heterocycloalkyl substituted with 0-3 R27, aralkyl substituted with 0-3 R27, alkaryl substituted with 0-3 R27, heterocycle substituted with 0-3 R27, and an electron, provided that when one of R22 or R23 is an electron, then the other is also an electron;
R25 and R26 are each independently selected from the group: a bond to Lnxe2x80x2, xe2x80x94OH, C1-C10 alkyl substituted with 0-3 R27, C1-C10 alkyl substituted with 0-3 R27, aryl substituted with 0-3 R27, cycloalkyl substituted with 0-3 R27, heterocycloalkyl substituted with 0-3 R27, aralkyl substituted with 0-3 R27, alkaryl substituted with 0-3 R27, and heterocycle substituted with 0-3 R27;
R27 is independently selected at each occurrence from the group: a bond to Lnxe2x80x2, xe2x95x90O, F, Cl, Br, I, CF3, CN, xe2x80x94CO2R28, xe2x80x94C(xe2x95x90O)R28, xe2x80x94C(xe2x95x90O)N(R28)2, xe2x80x94CHO, xe2x80x94CH2OR28, xe2x80x94OC(xe2x95x90O)R28, xe2x80x94OC(xe2x95x90O)OR28a, xe2x80x94OR28, xe2x80x94OC(xe2x95x90O)N(R28)2, xe2x80x94NR29C(xe2x95x90O)R28, xe2x80x94NR29C(xe2x95x90O)OR28a, xe2x80x94NR29C(xe2x95x90O)N(R28)2, xe2x80x94NR29SO2N(R28)2, xe2x80x94NR29SO2R28a, xe2x80x94SO3H, xe2x80x94SO2R28a, xe2x80x94SR28, xe2x80x94S(xe2x95x90O)R28a, xe2x80x94SO2N(R28)2, xe2x80x94N(R28)2, xe2x80x94NHC(xe2x95x90NH)NHR28, xe2x80x94C(xe2x95x90NH)NHR28, xe2x95x90NOR28, NO2, xe2x80x94C(xe2x95x90O)NHOR28, xe2x80x94C(xe2x95x90O)NHNR28R28a, xe2x80x94OCH2CO2H, 2-(1-morpholino)ethoxy, C1-C5 alkyl, C2-C4 alkenyl, C3-C6 cycloalkyl, C3-C6 cycloalkylmethyl, C2-C6 alkoxyalkyl, aryl substituted with 0-2 R28, and a 5-10-membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O;
R28, R28a, and R29 are independently selected at each occurrence from the group: a bond to Lnxe2x80x2, H, C1-C6 alkyl, phenyl, benzyl, C1-C6 alkoxy, halide, nitro, cyano, and trifluoromethyl;
Z is selected from the group: 
xe2x80x83wherein,
A10 is NR41 or xe2x80x94C(R41)xe2x95x90CHxe2x80x94;
R37 is selected from the group: C(xe2x95x90O)xe2x80x94R42, CHxe2x95x90CR43C(xe2x95x90O)xe2x80x94R42, CH2C(xe2x95x90O)xe2x80x94R42, and CH2CH2C(xe2x95x90O)xe2x80x94R42;
R38 is selected from the group: SR44, SCH2R44, and S(xe2x95x90O)R44;
R39 is selected from the group: C1-C10 alkyl substituted with 0-3 R44, and C1-C10 alkoxy substituted with 0-3 R44;
R40 is C(xe2x95x90O)xe2x80x94R42;
R41 is CH2C(xe2x95x90O)N(CH3)CH2CH2C6H5;
R42 is a bond to Lnxe2x80x2;
R43 is selected from the group: H and C1-C3 alkyl;
R44 is phenyl substituted with 0-4 R45;
R45 is independently selected at each occurrence from the group: C1-C4 alkyl, OR46, C(xe2x95x90O)OR46, xe2x80x94Cl, xe2x80x94Br, xe2x80x94F, and N(R46)2;
R46 is independently selected at each occurrence from the group: H, and C1-C10 alkyl; and
e indicates the position of an optional double bond;
and pharmaceutically acceptable salts thereof.
[23] In an embodiment, the present invention provides a method according to embodiment [22] wherein:
We is selected from the group: 
xe2x80x83wherein,
A1 is N, Cxe2x80x94OH, or CH;
A2 and A3 are CH;
A4 is CR3;
A5 is O;
A6 is O or CH2;
R4 is independently selected from the group: xe2x80x94F, xe2x80x94Cl, xe2x95x90O, xe2x80x94N(R6)(R7), and xe2x80x94CF3;
R5 is independently selected from the group: xe2x80x94F, xe2x80x94Cl, xe2x80x94CF3, C1-C3 alkyl, C1-C3 alkoxy, and methylenedioxy;
X is O, CH2 or CHxe2x95x90CH;
R8, R9, R10 and R11 are independently selected at each occurrence from the group: a bond to Lnxe2x80x2, H, C1-C5 alkyl, and C1-C5 alkoxy;
or alternatively, R8 and R9 or R10 and R11 may be taken together to form a 3-6 membered cycloalkyl;
Ch is selected from the group: 
xe2x80x83wherein:
Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are independently selected at each occurrence from the group: NR22, NR22R23, S, SH, OH;
E is a bond, CH, or a spacer group selected from the group: C1-C10 alkyl substituted with 0-3 R27, aryl substituted with 0-3 R27, cycloalkyl substituted with 0-3 R27, and heterocycle substituted with 0-3 R27;
Ea is CH or a C3-C6 carbocycle;
R22 and R23 are each independently selected from the group: a bond to Lnxe2x80x2, hydrogen, C1-C10 alkyl substituted with 0-3 R27, aryl substituted with 0-3 R27, heterocycle substituted with 0-3 R27, and an electron, provided that when one of R22 or R23 is an electron;
R27 is independently selected at each occurrence from the group: a bond to Lnxe2x80x2, xe2x95x90O, F, Cl, Br, I, xe2x80x94CF3, xe2x80x94CN, xe2x80x94CO2R28, xe2x80x94C(xe2x95x90O)R28, xe2x80x94C(xe2x95x90O)N(R28)2, xe2x80x94CH2OR28, xe2x80x94OC(xe2x95x90O)R28, xe2x80x94OC(xe2x95x90O)OR28a, xe2x80x94OR28, xe2x80x94OC(xe2x95x90O)N(R28)2, xe2x80x94NR29C(xe2x95x90O)R28, xe2x80x94NR29C(xe2x95x90O)OR28a, xe2x80x94NR29C(xe2x95x90O)N(R28)2, xe2x80x94NR29SO2N(R28)2, xe2x80x94NR29SO2R28a, xe2x80x94SO3H, xe2x80x94SO2R28a, xe2x80x94SR28, xe2x80x94S(xe2x95x90O)R28a, xe2x80x94SO2N(R28)2, xe2x80x94N(R28)2, xe2x80x94NHC(xe2x95x90NH)NHR28, xe2x80x94C(xe2x95x90NH)NHR28, xe2x95x90NOR28, NO2, xe2x80x94C(xe2x95x90O)NHOR28, xe2x80x94C(xe2x95x90O)NHNR28R28a, xe2x80x94OCH2CO2H, and 2-(1-morpholino)ethoxy;
R28, R28a, and R29 are independently selected at each occurrence from the group: a bond to Lnxe2x80x2, H, and C1-C6 alkyl;
R39 is selected from the group: C1-C10 alkyl substituted with 0-1 R44, and C1-C10 alkoxy substituted with 0-1 R44;
R43 is H; and
R46 is independently selected at each occurrence from the group: H, and C1-C5 alkyl.
[24] In an embodiment, the present invention provides a reagent of Embodiment [22] wherein:
R1 is selected from the group: H, xe2x80x94C(xe2x95x90NH)NH2, C1-C6 alkyl substituted with 0-2 R4, C1-C6 alkoxy substituted with 0-2 R4, aryl substituted with 0-2 R5, and heterocycle substituted with 0-2 R5;
R3 is xe2x80x94H, xe2x80x94OH or C1-C3 alkoxy;
or alternatively, R1 and R3 can be taken together with the atoms to which they are attached to form a fused phenyl ring substituted with 0-2 R5;
R4 is independently selected from the group: xe2x95x90O, and xe2x80x94N(R6)(R7);
R5 is independently selected from the group: xe2x80x94F, C1-C3 alkyl, C1-C3 alkoxy, and methylenedioxy;
X is O, CH2 or CHxe2x95x90CH;
R8, R9, R10 and R11 are independently selected at each occurrence from the group: a bond to Lnxe2x80x2, H, and C1-C3 alkyl;
or alternatively, R8 and R9 or R10 and R11 may be taken together to form a 3-6 membered cycloalkyl;
W1 is O;
M1 is selected from the group: phenyl substituted with 0-1 R12, heterocycle substituted with 0-1 R12, benzophenone substituted with 0-1 R12, and diphenylether substituted with 0-1 R12;
R12 is independently selected from the group: a bond to Lnxe2x80x2, xe2x80x94COOR13, C1-C5 alkyl substituted with 0-1 R14, and C1-C5 alkoxy substituted with 0-1 R14;
M2 is selected from the group: aryl substituted with 0-1 R19, cycloalkyl substituted with 0-3 R19, and heterocycle substituted with 0-1 R19;
Ch is selected from: 
wherein,
Q1 and Q4 are SH;
Q2 and Q3 are NR22;
E is independently selected from the group: CHR27, CH2CHR27, CH2CH2CHR27, and CHR27C(xe2x95x90O);
R22 is selected from the group: H, C1-C6 alkyl substituted with 0-1 R27; and
R27 are independently selected from H and a bond to Lnxe2x80x2,
and, 
wherein
E is a bond;
Q2 is NHR23, wherein R23 is heterocycle substituted with R27, wherein the heterocycle is selected from pyridine and thiazole, R27 is selected from C(xe2x95x90O)NHR28 and C(xe2x95x90O)R28, and R28 is a bond to Lnxe2x80x2;
A10 is NR41;
R39 is C1-C10 alkoxy substituted with 1 R44; and
R45 is independently selected at each occurrence from the group: C1-C4 alkyl, OH, C(xe2x95x90O)OH, xe2x80x94Cl, xe2x80x94F, and NH2.
[25] In an embodiment, the present invention provides a method of embodiment [22] wherein the reagent is selected from the group:
4-ethyl-2-(4-fluorophenyl)-[5-[5,5-dimethyl-6-[[6-[[[(2-sulfonylphenyl)methylene]hydrazino]-3-pyridinyl]carbonyl]amino]hexyl]oxy]phenol;
4-ethyl-2-(4-fluorophenyl)-[5-[4-[[6-[[[(2-sulfonylphenyl)methylene]hydrazino]-3-pyridinyl]carbonyl]amino]butyl]oxy]phenol;
2-[[[5-[[(6-[(4,6-diphenyl-2-pyridinyl)oxy]-1-hexanamino]carbonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonic acid;
2-[[[5-[[2,2-dimethyl-6-[(6-fluorophenyl-4-phenyl-2-pyridinyl)oxy]-1-hexanamino]carbonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonic acid;
2-[[[5-[[N-[6-[(6-(4-fluorophenyl)-4-phenyl-2-pyridinyl)oxy]-hexanoyl]-tyrosine-O-[3-propanamino]]carbonyl]-2-pyridinyl]hydrazono]-methyl]-benzenesulfonic acid;
2-[[[5-[[N-[6-[(4,6-diphenyl-2-pyridinyl)oxy]-hexanoyl]-tyrosine-O-[3-propanamino]]carbonyl]-2-pyridinyl]hydrazono]-methyl]-benzenesulfonic acid;
2-[[[5-[[N-[6-[(4-(3,4-methylenedioxyphenyl)-6-phenyl-2-pyridinyl)oxy]-hexanoyl]-tyrosine-O-[3-propanamino]]-carbonyl]-2-pyridinyl]hydrazono]-methyl]-benzenesulfonic acid;
2-[[[5-[[alpha-N-[6-[(4,6-diphenyl-2-pyridinyl)oxy]-hexanoyl]-lysine-epsilon-N-amino]carbonyl]-2-pyridinyl]hydrazono]-methyl]-benzenesulfonic acid;
4-ethyl-2-(4-fluorophenyl)-5-[(5,5-dimethyl-6-aminohexyl)oxy]phenol N-[4-(carboxy)benzyl]-N,Nxe2x80x2-bis[2-thioethyl]-glycinamide Conjugate;
Benzenesulfonic Acid, 2-[[[5-[[[6-[(4,6-diphenyl-2-pyridinyl)oxy]2,2-dimethyl-1-hexyl]aza]carbonyl]-2-pyridinyl]hydrazono]methyl];
2-[[[5-[[[[6-[(4,6-Diphenyl-2-pyridinyl)oxy]-hexanoyl]-4-sulfonamidyl]benzylamino]carbonyl]-2-pyridinyl]-hydrazono]methyl]-benzenesulfonic acid;
4-ethyl-2-(4-fluorophenyl)-[5-[6,6-dimethyl-7-[[6-[[[(2-sulfonylphenyl)methylene]hydrazino]-3-pyridinyl]carbonyl]amino]heptyl]oxy]phenol;
4-ethyl-2-(5-pyrazolyl)-[5-[5,5-dimethyl-6-[[6-[[[(2-sulfonylphenyl)methylene]hydrazino]-3-pyridinyl]carbonyl]amino]hexyl]oxy]phenol;
the Conjugate Between 2-[6-[(4,6-Diphenyl-2-pyridinyl)-oxy]pentyl]-6-(8-amino-5-aza-4-oxooctyloxy)-benzenepropanoic Acid and Benzenesulfonic Acid, 2-[[[5-[[(2,5-Dioxo-1-pyrrolidinyl)oxy]carbonyl]-2-pyridinyl]-hydrazono]methyl];
the Conjugate Between 6-(11-Amino-3,6,9-trioxaundecyl oxy)-2-[5-[(5-oxo-1-(2-propenyl)-5,6,7,8-tetrahydro-2-naphthalenyl)oxy]pentyloxy]benzenepropanoic Acid and Benzenesulfonic Acid, 2-[[[5-[[(2,5-Dioxo-1-pyrrolidinyl)oxy]carbonyl]-2-pyridinyl]-hydrazono]methyl];
4-ethyl-2-(4-fluorophenyl)-[5-[6,6-dimethyl-7-[[6-[[[phenylmethylene]hydrazino]-3-pyridinyl]carbonyl]amino]heptyl]oxy]phenol;
N-((6-((1-aza-2-phenylvinyl)amino)(3-pyridyl))sulfonyl)-3-(1-((N-(2-phenylethyl)carbamoyl)methyl)-5-(phenylmethoxy)indol-3-yl)prop-2-enamide;
propyl 3-((7-(3-(6-ethyl-4-(4-fluorophenyl)-3-hydroxyphenoxy)propoxy)-8-propylchroman-2-yl)carbonylamino)propanoate, 2-(2-aza-2-((5-carbamoyl(2-pyridyl)amino)vinyl)benzenesulfonic acid;
3-((7-(-(6-ethyl-4-(4-fluorophenyl)-3-hydroxyphenoxy)propoxy)-8-propylchroman-2-yl)carbonylamino)propyl-2-methylpropanoate, 2-(2-aza-2((5-carbamoyl-(2-pyridyl)amino)vinyl)benzenesulfonic acid;
N-(3-((7-(3-(6-ethyl-4-(4-fluorophenyl)-3-hydroxyphenoxy)propoxy)-8-propylchroman-2-yl)carbonylamino)propyl)-2-methylpropanamide, 2-(2-aza-2-((5-carbamoyl-(2-pyridyl))amino)vinyl)benzenesulfonic acid;
2-(2-aza-2-((5-(N-(6-(6-ethyl-3-hydroxy-4-(1-methylpyrazol-5-yl)phenoxy)-22-dimethylhexyl)carbamoyl)(2-pyridyl))amino)vinyl)benzenesulfonic acid;
2-(2-aza-2-((5-(N-(6-(6-ethyl-3-hydroxy-4-(1-methylpyrazol-5-yl)phenoxy)-2,2-dimethylhexyl)carbamoyl)(2-pyridyl))amino)vinyl)benzenesulfonic acid;
2-(2-aza-2-((5-((3-((6-ethyl-4-(4-fluorophenyl)-3-hydroxyphenoxy)methyl)piperidyl)carbonyl)(2-pyridyl))amino)vinyl)benzenesulfonic acid;
2-(((4-(N-(6-(4,6-Diphenyl(2-pyridyloxy))-2,2-dimethylhexyl)carbamoyl)phenyl)methyl)(2-sulfanylethyl)amino)-N-(2-sulfanylethyl)ethanamide;
2-(2-Aza-2-((5-(N-(3-(2-(2-(3-(5-(4-(5-(4,6-diphenyl-(2-pyridyloxy))-1,1-dimethylpentyl)(1,2,3,5-tetraazolyl))pentanoylamino)propoxy)ethoxy)ethoxy)propyl)carbamoyl)(2-pyridyl))amino)vinyl)benzenesulfonic Acid;
2-(2-Aza-2-((5-(N-(3-(2-(2-(3-(5-(5-(5-(4,6-diphenyl-(2-pyridyloxy))-1,1-dimethylpentyl)(1,2,3,4-tetraazolyl))pentanoylamino)propoxy)ethoxy)ethoxy)propyl)carbamoyl)(2-pyridyl))amino)vinyl)benzenesulfonic Acid;
2-(2-Aza-2-((5-(N-(2-(2-(2-(2-(2-(2-(2-(2-(5-(4-(5-(4,6-diphenyl-(2-pyridyloxy))-1,1-dimethylpentyl)(1,2,3,5-tetraazolyl))pentanoylamino)ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)(2-pyridyl))amino)vinyl)benzenesulfonic Acid;
2-(2-Aza-2-((5-(N-(5-(4-(5-(4,6-diphenyl-(2-pyridyloxy))-1,1-dimethylpentyl)(1,2,3,5-tetraazolyl))pentanoylamino)-1-(6-deoxy-xcex2-cyclodextryl)carbamoyl)pentyl)carbamoyl)(2-pyridyl))amino)vinyl)benzenesulfonic Acid;
2-(2-Aza-2-((5-(N-(3-(2-(2-(3-(2-(5-(5-(4,6-diphenyl-(2-pyridyloxy))-1,1-dimethylpentyl) (1,2,3,4-tetraazolyl))acetylamino)propoxy)ethoxy)ethoxy)propyl)-carbamoyl)(2-pyridyl))amino)vinyl)benzenesulfonic Acid;
2-(2-Aza-2-((5-(N-(3-(2-(2-(3-(2-(4-(5-(4,6-diphenyl-(2-pyridyloxy))-1,1-dimethylpentyl)(1,2,3,5-tetraazolyl))acetylamino)propoxy)ethoxy)ethoxy)propyl)-carbamoyl)(2-pyridyl))amino)vinyl)benzenesulfonic Acid;
3-(6-(3-(N-(5-((6-((1-Aza-2-(sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)-5-(N-(xcfx89-methoxypolyethylene-(750)glycoxyethyl)carbamoyl)pentyl) carbamoyl)propoxy)2(5-(4,6-diphenyl-(2-pyridyloxy))pentyloxy)phenyl)propanoic Acid;
3-(6-(3-(N-(3-(2-(2-(3-((6-((1-Aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)propoxy)ethoxy)ethoxy)propyl)-carbamoyl)propoxy)2-(5-(4,6-diphenyl-(2-pyridyloxy))pentyloxy)phenyl)propanoic Acid;
3-(6-(3-(N-(5-((6-((1-Aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)-5-(N-(2,3,4,5,6-pentahydroxyhexyl)carbamoyl)pentyl)carbamoyl)propoxy)2-(5-(4,6-diphenyl-(2-pyridyloxy))pentyloxy)phenyl)propanoic Acid;
3-(6-(3-(N-(3-((6-((1-Aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino) propyl)carbamoyl)propoxy)-2-(5-(5-oxo-1-prop-2-enyl-(2-6,7,8-trihydronaphthyloxy))pentyloxy)phenyl)propanoic Acid;
3-(6-(3-N-(2-(2-(2-(2-(2-(2-(2-(2-((6-((1-Aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)propoxy)-2-(5-(5-oxo-1-prop-2-enyl-(2-6,7,8-trihydronaphthyloxy))pentyloxy)phenyl)propanoic Acid;
3-(6-(3-N-(5-((6-((1-Aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)-5-(N-(2,3,4,5,6-pentahydroxyhexyl)carbamoyl)pentyl)carbamoyl)propoxy-2-(5-(5-oxo-1-prop-2-enyl-(2-6,7,8-trihydronaphthyloxy))pentyloxy)phenyl)propanoic Acid;
3-(6-(3-N-(5-((6-((1-Aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)-5-(N-(6-deoxy-xcex2-cyclodextryl)carbamoyl)pentyl)carbamoyl)propoxy-2-(5-(5-oxo-1-prop-2-enyl-(2-6,7,8-trihydronaphthyloxy))pentyloxy)phenyl)propanoic Acid;
3-(6-(3-(N-(3-((6-((1-Aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))-Gly-Lys-Lys-Lys)aminopropyl)carbamoyl)propoxy)-2-(5-(5-oxo-1-prop-2-enyl-(2-6,7,8-trihydronaphthyloxy))pentyloxy)phenyl)propanoic Acid;
2-Sulfobenzaldehyde (E)-N-[3-(6-Hydrazinonicotinamido)propyl]-3-[6-[[(2,6-dichlorophenyl)thio]methyl]-3-(2-phenylethoxy)-2-pyridinyl]-2-propenamide Hydrazone;
2-Sulfobenzaldehyde (E)-N-[3-(6-Hydrazinonicotinamido)propyl]-3-[6-[(phenylthio)methyl]-3-(2-phenylethoxy)-2-pyridinyl]-2-propenamide Hydrazone;
2-Sulfobenzaldehyde (E)-N-[3-(6-Hydrazinonicotinamido)propyl]-3-[6-[[(2-chlorophenyl)thio]methyl]-3-(2-phenylethoxy)-2-pyridinyl]-2-propenamide Hydrazone;
2-Sulfobenzaldehyde (E)-N-[3-(6-Hydrazinonicotinamido)propyl]-3-[6-[[(2,6-dimethylphenyl)thio]methyl]-3-(2-phenylethoxy)-2-pyridinyl]-2-propenamide Hydrazone;
2-Sulfobenzaldehyde (E)-N-[3-(6-Hydrazinonicotinamido)propyl]-3-[6-[[(2,3,5,6-tetrafluorophenyl)thio]methyl]-3-(2-phenylethoxy)-2-pyridinyl]-2-propenamide Hydrazone;
2-Sulfobenzaldehyde (E)-N-[3-(6-Hydrazinonicotinamido)propyl]-3-[6-[[(4-hydroxyphenyl)thio]methyl]-3-(2-phenylethoxy)-2-pyridinyl]-2-propenamide Hydrazone;
2-Sulfobenzaldehyde (E)-N-[2-(6-Hydrazinonicotinamido)ethyl]-3-[6-[[(2,6-dichlorophenyl)thio]methyl]-3-(2-phenylethoxy)-2-pyridinyl]-2-propanamide Hydrazone;
2-Sulfobenzaldehyde N-[3-(6-Hydrazinonicotinamido)propyl]-1-[3-([1,1xe2x80x2-biphenyl]-4-ylmethyl)-2H-1-benzopyran-7-yl]-cyclopentanecarboxamide Hydrazone;
2-Sulfobenzaldehyde 6-[5-(6-Hydrazinonicotinamido)pentyloxy]-5-(2-propenyl)-1,2,3,4-tetrahydronaphthalen-1-one Hydrazone;
2-Sulfobenzaldehyde 6-[6-(6-Hydrazinonicotinamido)hexyloxy]-5-(2-propenyl)-1,2,3,4-tetrahydronaphthalen-1-one Hydrazone;
2-Sulfobenzaldehyde 6-[6-(6-Hydrazinonicotinamido)-4,4-dimethylpentyloxy]-5-(2-propenyl)-1,2,3,4-tetrahydronaphthalen-1-one Hydrazone;
2-Sulfobenzaldehyde 6-[6-(6-Hydrazinonicotinamido)-5,5-dimethylhexyloxy]-5-(2-propenyl)-1,2,3,4-tetrahydronaphthalen-1-one Hydrazone;
2-Sulfobenzaldehyde 6-[4-(6-Hydrazinonicotinamido)butoxy]-5-(2-propenyl)-1,2,3,4-tetrahydronaphthalen-1-one Hydrazone;
2-Sulfobenzaldehyde 6-[3-(6-Hydrazinonicotinamido)propoxy]-5-(2-propenyl)-1,2,3,4-tetrahydronaphthalen-1-one Hydrazone;
2-Sulfobenzaldehyde 6-[2-(6-Hydrazinonicotinamido)ethoxy]-5-(2-propenyl)-1,2,3,4-tetrahydronaphthalen-1-one Hydrazone;
2-[[[5-[[2,2-Dimethyl-6-[(4-(3,4-methylenedioxyphenyl)-6-phenyl-2-pyridinyl)oxy]-1-hexanamino]carbonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonic acid;
N-[2,2-Dimethyl-6-[(4-(3,4-methylenedioxyphenyl)-6-phenyl-2-pyridinyl)oxy]-hexyl]-bis-S-(1-ethoxyethylmercapto-acetyl)pentanoate;
2-[[[5-[[N-[6-[(4,6-diphenyl-2-pyridinyl)oxy]-hexanoyl]-glycine-alpha-amino]carbonyl]-2-pyridinyl]hydrazono]-methyl]-benzenesulfonic acid;
2-Acetyl-4-ethyl-[5-[6-[[6-[[[(2-sulfonylphenyl)methylene]hydrazino]-3-pyridinyl]carbonyl]amino]hexyl]oxy]phenol;
2,4-Diethyl-[5-[5,5-dimethyl-6-[[6-[[[(2-sulfonylphenyl)methylene]hydrazino]-3-pyridinyl]carbonyl]amino]hexyl]oxy]phenol;
3-(4-(5-(4,6-diphenyl-(2-pyridyloxy))pentyloxy)-3-ethoxyphenyl)-N-((6-hydrazino(3-pyridyl))sulfonyl)prop-2-enamide;
2-((6-((1-aza-2-(2-sulfophenyl)vinyl)-amino)(3-pyridyl))carbonyl)-7-(5-(4,6-diphenyl(2-pyridyl-oxy))pentyloxy)-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid;
2-((6-((1-aza-2-(2-sulfophenyl)vinyl)-amino)(3-pyridyl)carbonylamino)-3-(4-(5-(4,6-diphenyl(2-pyridyloxy))pentyloxy)phenyl)propanoic acid;
2-((6-((1-aza-2-(2-sulfophenyl)vinyl)-amino)(3-pyridyl)carbonylamino)-3-(2-(5-(4,6-diphenyl(2-pyridyloxy))pentyloxy)phenyl)propanoic acid;
3-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl)carbonylamino)-3-(N-(6-(4,6-diphenyl(2-pyridyloxy))-2,2-dimethylhexyl)carbamoyl)propanoic acid;
2-(2-aza-2-((5-(N-(3-(2-(2-(3-((1-((N-methyl-N-(2-phenylethyl)carbamoyl)methyl)-5-(phenylmethoxy)-indol-2-yl)carbonylamino)propoxy)ethoxy)ethoxy)propyl)-carbamoyl)(2-pyridyl))amino)vinyl)benzenesulfonic acid;
2-(2-((6-((1-aza-2-(2-sulfophenyl)vinyl)-amino)(3-pyridyl)carbonylamino)-3-carboxypropanoylamino)-3-(2-(5-(4,6-diphenyl(2-pyridyloxy))pentyloxy)phenyl)propanoic acid;
2-(2-aza-2-((5-(N-(2-(N-(3-(2-(2-(3-(2-(2,5-dioxoimidazolidin-4-yl)acetylamino)-propoxy)ethoxy)ethoxy)-propyl)carbamoyl)-1-(N-(6-(4,6-diphenyl(2-pyridyloxy))-2,2-dimethylhexyl)carbamoyl)-ethyl)carbamoyl(2-pyridyl))amino)-vinyl)benzenesulfonic acid;
6-((6-((1-aza-2-(2-sulfophenyl)-vinyl)amino)(3-pyridyl)carbonylamino)-2-((1-((N-methyl-N-(2-phenylethyl)carbamoyl)methyl)-5-(phenylmethoxy)indol-2-yl)carbonylamino)hexanoic acid;
1-(3-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)-(3-pyridyl)carbonylamino)-3-(N-(6-(4,6-diphenyl(2-pyridyloxy))-2,2-dimethylhexyl)carbamoyl)propanoylamino)-ethane-1,2-dicarboxylic acid;
1-(2-(3-((6-((1-aza-2-(2-sulfophenyl)vinyl)-amino)(3-pyridyl)carbonylamino)-3-(N-(6-(4,6-diphenyl(2-pyridyloxy))-2,2-dimethylhexyl)carbamoyl)propanoylamino)-3-carboxypropanoylamino)ethane-1,2-dicarboxylic acid;
2-(2-aza-2-((5-(N-(1-(N-(6-(4,6-diphenyl(2-pyridyloxy))-2,2-dimethylhexyl)carbamoyl)-2-(3-(((4,5,6-trihydroxy-3-(hydroxymethyl)(2-oxanyl))amino)carbonylamino)-propanoylamino)ethyl)carbamoyl(2-pyirdyl))amino)vinyl)-benzenesulfonic acid;
2-(2-aza-2-((5-((6-(4-benzo[d] 1,3-dioxolan-5-yl-6-phenyl(2-pyridyloxy))-2,2-dimethylhexanoyl-amino)sulfonyl)-(2-pyridyl))amino)vinyl)benzenesulfonic acid;
6-(4-benzo[d] 1,3-dioxolan-5-yl-6-phenyl(2-pyridyloxy))-N-(1-(N-((6-hydrazino(3-pyridyl))sulfonyl)cabamoyl)-2-(4-hydroxyphenyl)ethyl)-2,2-dimethylhexanamide;
4-(4,6-diphenyl(2-pyridyloxy))-N-(1-(N-(1-(N-((6-hydrazino(3-pyridyl))sulfonyl)cabamoyl)-2-(4-hydroxyphenyl)ethyl)-carbamoyl)-isopropyl)butanamide;
3-(4-(3-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)-propoxy)phenyl)-2-(2,2-dimethyl-6-(5-oxo-1-prop-2-enyl(2-6,7,8-trihydronaphthyloxy))hexanoylamino)propanoic acid;
3-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl)carbonylamino)-3-(N-(6-(4-benzo[d] 1,3-dioxolan-5-yl-6-phenyl(2-pyridyloxy))-2,2-dimethylhexyl)carbamoyl)propanoic acid;
2-(2-aza-2-((5-(N-(1-(N-(6-(4-benzo[d] 1,3-dioxolan-5-yl-6-phenyl(2-pyridyloxy))-2,2-dimethyl-hexyl)carbamoyl)-2-(4-hydroxyphenyl)ethyl)carbamoyl(2-pyridyl))amino)vinyl)-benzenesulfonic acid;
2-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl)carbonylamino)-2-(2,2-dimethyl-6-(5-oxo-1-prop-2-enyl(2-6,7,8-trihydronaphthyloyy))hexanoylamino)acetic acid;
2-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl)carbonylamino)-2-(2,2-dimethyl-6-(5-oxo-1-prop-2-enyl(2-6,7,8-trihydronaphthyloxy))hexanoylamino)acetic acid;
3-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl)carbonylamino)-3-(N-(6-(6-ethyl-3-hydroxy-4-phenylphenoxy)-2,2-dimethylhexyl)carbamoyl)propanoic acid;
2-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl)carbonylamino)-2-(6-(4-benzo[d] 1,3-dioxolan-5-yl-6-phenyl(2-pyridyloxy))-2,2-dimethylhexanoylamino)acetic acid;
2-(2-aza-2-((5-(N-(5-((3-((N-(6-(4,6-diphenyl(2-pyridyloxy))-2,2-dimethylhexanoylamino)-3-(4-hydroxyphenyl)propanoylamino)-1-(N-(2,3,4,5,6-pentahydroxyhexyl)carbamoyl)pentyl)carbamoyl(2-pyridyl))amino)vinyl)benzenesulfonic acid;
2-(2-aza-2-((5-(N-(5-((3-((N-(6-(4-benzo[d] 1,3-dioxolan-5-yl-6-phenyl(2-pyridyloxy))-2,2-dimethylhexyl)-carbamoyl)-2-(N-(2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-ethyl)carbamoyl(2-pyridyl))amino)vinyl)benzenesulfonic acid;
2-(2-aza-2-((5-(N-(5-((3-((N-(6-(4-benzo[d] 1,3-dioxolan-5-yl-6-phenyl(2-pyridyloxy))-2,2-dimethylhexyl)carbamoyl)amino)phenyl)carbonylamino)-1-(N-(2,3,4,5,6-pentahydroxyhexyl)carbamoyl)pentyl)carbamoyl(2-pyridyl))amino)vinyl)benzenesulfonic acid;
2-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)-3-(N-(6-(4-benzo[d] 1,3-dioxolan-5-yl-6-phenyl(2-pyridyloxy))-2,2-dimethylhexyl)carbamoyl)-propanoylamino)-3-carboxypropanoylamino)-3-carboxypropanoylamino)-ethane-1,2-dicarboxylic acid;
2-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)-3-(2-(5-(4-benzo[d] 1,3-dioxolan-5-yl-6-phenyl(2-pyridyloxy))pentyloxy)phenyl)propanoic acid; and
2-({2-[((2S)-2-[Bis(carboxymethyl)amino]-3-{4-[({[3-(2-{2-[3-(5-{5-[5-(4,6-diphenyl(2-pyridyloxy))-1,1-dimethylpentyl](5H-1,2,3,4-tetraazolyl)}pentanoylamino)propoxy]ethoxy}ethoxy)propyl]amino}thioxomethyl)amino]phenyl}propyl)(carboxymethyl)amino]ethyl}-(carboxymethyl)amino)acetic acid.
In an embodiment, the present invention provides a method according to any one of embodiments [1-21] wherein the radiolabeled LTB4 binding agent is selected from the group consisting of:
99mTc(tricine)(TPPTS)(4-ethyl-2-(4-fluorophenyl)-[5-[5,5-dimethyl-6-[[[6-diazenido-3-pyridinyl]carbonyl]amino]hexyl]oxy]phenol);
99mTc(tricine)(TPPDS)(4-ethyl-2-(4-fluorophenyl)-[5-[5,5-dimethyl-6-[[[6-diazenido-3-pyridinyl]carbonyl]amino]hexyl]oxy]phenol);
99mTc(tricine)(TPPMS)(4-ethyl-2-(4-fluorophenyl)-[5-[5,5-dimethyl-6-[[[6-diazenido-3-pyridinyl]carbonyl]amino]hexyl]oxy]phenol);
99mTc(tricine)(3-sulfonatopyridine))(4-ethyl-2-(4-fluorophenyl)-[5-[5,5-dimethyl-6-[[[6-diazenido-3-pyridinyl]carbonyl]amino]hexyl]oxy]phenol);
99mTc(tricine)(TPPTS)(4-ethyl-2-(4-fluorophenyl)-[5-[4-[[[6-diazenido-3-pyridinyl]carbonyl]amino]butyl]oxy]phenol);
99mTc(tricine)(TPPTS)(2-[[5-[[(6-[(4,6-diphenyl-2-pyridinyl)oxy]-1-hexanamino]carbonyl]-2-pyridinyl]diazenido]);
99mTc(tricine)(TPPTS)(2-[[5-[[2,2-dimethyl-6-[(6-fluorophenyl-4-phenyl-2-pyridinyl)oxy]-1-hexanamino]carbonyl]-2-pyridinyl]diazenido]);
99mTc(tricine)(TPPTS)(2-[[5-[[N-[6-[(4,6-diphenyl-2-pyridinyl)oxy]-hexanoyl]-tyrosine-O-[3-propanamino]]carbonyl]-2-pyridinyl]diazenido]);
99mTc(tricine)(TPPTS)(2-[[[5-[[N-[6-[(4-(3,4-methylenedioxyphenyl)-6-phenyl-2-pyridinyl)oxy]-hexanoyl]-tyrosine-O-[3-propanamino]]-carbonyl]-2-pyridinyl]diazenido]);
99mTc(tricine)(TPPTS)(2-[[[5-[[alpha-N-[6-[(4,6-diphenyl-2-pyridinyl)oxy]-hexanoyl]-lysine-epsilon-N-amino]carbonyl]-2-pyridinyl]diazenido]);
99mTc(tricine)(TPPTS)(2-[[[5-[[[6-[(4,6-diphenyl-2-pyridinyl)oxy]-2,2-dimethyl-1-hexyl]aza]carbonyl]-2-pyridinyl]diazenido]);
99mTc(tricine)(TPPTS)(4-ethyl-2-(4-fluorophenyl)-[5-[6,6-dimethyl-7-[[6-[[6-diazenido]-3-pyridinyl]carbonyl]amino]heptyl]oxy]phenol);
99mTc(tricine)(TPPTS)(2-[6-[(4,6-Diphenyl-2-pyridinyl)oxy]pentyl]-6-(8-[[[6-diazenido]-3-pyridinyl]carbonyl]amino-5-aza-4-oxooctyloxy)-benzenepropanoic Acid);
99mTc(tricine)(3-pyridinesulfonic acid))(2-[6-[(4,6-Diphenyl-2-pyridinyl)oxy]pentyl]-6-(8-[[[6-diazenido]-3-pyridinyl]carbonyl]amino-5-aza-4-oxooctyloxy)benzenepropanoic Acid);
99mTc(tricine)(3,5-pyridinedicarboxylic acid)(2-[6-[(4,6-Diphenyl-2-pyridinyl)oxy]pentyl]-6-(8-[[[6-diazenido]-3-pyridinyl]carbonyl]amino-5-aza-4-oxooctyloxy)benzenepropanoic Acid);
99mTc(tricine)(TPPTS)(6-(11-[[[6-diazenido]-3-pyridinyl]carbonyl]amino-3,6,9-trioxaundecyloxy)-2-[5-[(5-oxo-1-(2-propenyl)-5,6,7,8-tetrahydro-2-naphthalenyl)oxy]pentyloxylbenzenepropanoic Acid);
99mTc(tricine)(TFP)(6-(11-[[[6-diazenido]-3-pyridinyl]carbonyl]amino-3,6,9-trioxaundecyloxy)-2-[5-[(5-oxo-1-(2-propenyl)-5,6,7,8-tetrahydro-2-naphthalenyl)oxy]pentyloxy]benzenepropanoic Acid);
99mTc(tricine)(3,5-pyridinedicarboxylic acid)(6-(11-[[[6-diazenido]-3-pyridinyl]carbonyl]amino-3,6,9-trioxaundecyloxy)-2-[5-[(5-oxo-1-(2-propenyl)-5,6,7,8-tetrahydro-2-naphthalenyl)oxy]pentyloxy]benzenepropanoic Acid);
99mTc(tricine)(isonicotinic acid)(6-(11-[[[6-diazenido]-3-pyridinyl]carbonyl]amino-3,6,9-trioxaundecyloxy)-2-[5-[(5-oxo-1-(2-propenyl)-5,6,7,8-tetrahydro-2-naphthalenyl)oxy]pentyloxy]benzenepropanoic Acid);
99mTc(tricine)(nicotinic acid)(6-(11-[[[6-diazenido]-3-pyridinyl]carbonyl]amino-3,6,9-trioxaundecyloxy)-2-[5-[(5-oxo-1-(2-propenyl)-5,6,7,8-tetrahydro-2-naphthalenyl)oxy]pentyloxy]benzenepropanoic Acid);
99mTc(tricine)(3-pyridinesulfonic acid)(6-(11-[[[6-diazenido]-3-pyridinyl]carbonyl]amino-3,6,9-trioxaundecyloxy)-2-[5-[(5-oxo-1-(2-propenyl)-5,6,7,8-tetrahydro-2-naphthalenyl)oxy]pentyloxy]benzenepropanoic Acid);
99mTc(tricine)(hydroxyethylisonicotinamide)(6-(11-[[[6-diazenido]-3-pyridinyl]carbonyl]amino-3,6,9-trioxaundecyloxy)-2-[5-[(5-oxo-1-(2-propenyl)-5,6,7,8-tetrahydro-2-naphthalenyl)oxy]pentyloxy]benzenepropanoic Acid);
99mTc(tricine)(4-methyl-5-imidazolemethanol)(6-(11-[[[6-diazenido]-3-pyridinyl]carbonyl]amino-3,6,9-trioxaundecyloxy)-2-[5-[(5-oxo-1-(2-propenyl)-5,6,7,8-tetrahydro-2-naphthalenyl)oxy]pentyloxy]benzenepropanoic Acid);
99mTc(tricine)(4-methyl-5-thiazoleethanol)(6-(11-[[[6-diazenido]-3-pyridinyl]carbonyl]amino-3,6,9-trioxaundecyloxy)-2-[5-[(5-oxo-1-(2-propenyl)-5,6,7,8-tetrahydro-2-naphthalenyl)oxy]pentyloxy]benzenepropanoic Acid);
99mTc(tricine)(pyridine)(6-(11-[[[6-diazenido]-3-pyridinyl]carbonyl]amino-3,6,9-trioxaundecyloxy)-2-[5-[(5-oxo-1-(2-propenyl)-5,6,7,8-tetrahydro-2-naphthalenyl)oxy]pentyloxy]benzenepropanoic Acid);
99mTc(tricine)(4-pyridylethylsulfonic acid)(6-(11-[[[6-diazenido]-3-pyridinyl]carbonyl]amino-3,6,9-trioxaundecyloxy)-2-[5-[(5-oxo-1-(2-propenyl)-5,6,7,8-tetrahydro-2-naphthalenyl)oxy]pentyloxy]benzenepropanoic Acid);
99mTc(tricine)(TPPTS)(N-((6-(diazenido)(3-pyridyl))sulfonyl)-3-(1-((N-(2-phenylethyl)carbamoyl)methyl)-5-(phenylmethoxy)indol-3-yl)prop-2-enamide);
99mTc(tricine)(TPPTS)((2-((5-carbamoyl(2-pyridyl)diazenido) ethyl 3-((7-(3-(6-ethyl-4-(4-fluorophenyl)-3-hydroxyphenoxy)propoxy)-8-propylchroman-2-yl)carbonylamino)propanoate);
99mTc(tricine)(TPPTS)(3-((7-(-(6-ethyl-4-(4-fluorophenyl)-3-hydroxyphenoxy)propoxy)-8-propylchroman-2-yl)carbonylamino)propyl-2-methylpropanoate, 2-(2((5-carbamoyl(2-pyridyl)diazenido);
99mTc(tricine)(TPPTS)(N-(3-((7-(3-(6-ethyl-4-(4-fluorophenyl)-3-hydroxyphenoxy)propoxy)-8-propylchroman-2-yl)carbonylamino)propyl)-2-methylpropanamide, 2-(2-((5-carbamoyl(2-pyridyl))diazenido);
99mTc(tricine)(TPPTS)(2-(2-((5-(N-(6-(6-ethyl-3-hydroxy-4-(1-methylpyrazol-5-yl)phenoxy)-2,2-dimethylhexyl)carbamoyl)(2-pyridyl))diazenido);
99mTc(tricine)(3-pyridinesulfonic acid)(2-(2-((5-(N-(6-(6-ethyl-3-hydroxy-4-(1-methylpyrazol-5-yl)phenoxy)-2,2-dimethylhexyl)carbamoyl)(2-pyridyl))diazenido);
99mTc(tricine)(TPPTS)(2-(2-((5-((3-((6-ethyl-4-(4-fluorophenyl)-3-hydroxyphenoxy)methyl)piperidyl)carbonyl)(2-pyridyl))diazenido);
99mTc(tricine)(TPPTS)(2-(2-((5-(N-(3-(2-(2-(3-(5-(5-(5-(4,6-diphenyl(2-pyridyloxy))-1,1-dimethylpentyl)(1,2,3,4-tetraazolyl))pentanoylamino)propoxy)ethoxy)ethoxy)propyl)carbamoyl)(2-pyridyl))diazenido);
99mTc(tricine)(TPPTS)(2-(2-((5-(N-(2-(2-(2-(2-(2-(2-(2-(2-(5-(4-(5-(4,6-diphenyl(2-pyridyloxy))-1,1-dimethylpentyl)(1,2,3,5-tetraazolyl))pentanoylamino)ethoxy)ethoxy)ethoxy)ethoxy) ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)(2-pyridyl))diazenido);
99mTc(tricine)(TPPTS)(2-(2-((5-(N-(5-(4-(5-(4,6-diphenyl(2-pyridyloxy))-1,1-dimethylpentyl)(1,2,3,5-tetraazolyl))pentanoylamino)-1-(6-deoxy-xcex2-cyclodextryl)carbamoyl)pentyl)carbamoyl)(2-pyridyl))diazenido);
99mTc(tricine)(TPPTS)(3-(6-(3-(N-(5-((6-(diazenido)(3-pyridyl))carbonylamino)-5-(N-((xcfx89-methoxypolyethylene(750)glycoxyethyl)carbamoyl)pentyl)carbamoyl)propoxy)2-(5-(4,6-diphenyl(2-pyridyloxy))pentyloxy)phenyl)propanoic Acid);
99mTc(tricine)(TPPTS)(3-(6-(3-(N-(3-(2-(2-(3-((6-(diazenido)(3-pyridyl))carbonylamino)propoxy)ethoxy)ethoxy)propyl)-carbamoyl)propoxy)2-(5-(4,6-diphenyl(2-pyridyloxy))pentyloxy)phenyl)propanoic Acid);
99mTc(tricine)(TPPTS)(3-(6-(3-(N-(5-((6-(diazenido)(3-pyridyl))carbonylamino)-5-(N-(2,3,4,5,6-pentahydroxyhexyl)carbamoyl)pentyl)carbamoyl)propoxy)2-(5-(4,6-diphenyl(2-pyridyloxy))pentyloxy)phenyl)propanoic Acid);
99mTc(tricine)(TFP)(3-(6-(3-(N-(5-((6-(diazenido)(3-pyridyl))carbonylamino)-5-(N-(2,3,4,5,6-pentahydroxyhexyl)carbamoyl)pentyl)carbamoyl)propoxy)2-(5-(4,6-diphenyl(2-pyridyloxy))pentyloxy)phenyl)propanoic Acid);
99mTc(tricine)(TPPTS)(3-(6-(3-(N-(3-((6-(diazenido)(3-pyridyl))carbonylamino)propyl)carbamoyl)propoxy)-2-(5-(5-oxo-1-prop-2-enyl(2-6,7,8-trihydronaphthyloxy))pentyloxy)phenyl)propanoic Acid);
99mTc(tricine)(TFP)(3-(6-(3-(N-(3-((6-(diazenido)(3-pyridyl))carbonylamino)propyl)carbamoyl)propoxy)-2-(5-(5-oxo-1-prop-2-enyl(2-6,7,8-trihydronaphthyloxy))pentyloxy)phenyl)propanoic Acid);
99mTc(tricine)(pyridine)(3-(6-(3-(N-(3-((6-(diazenido)(3-pyridyl))carbonylamino)propyl)carbamoyl)propoxy)-2-(5-(5-oxo-1-prop-2-enyl(2-6,7,8-trihydronaphthyloxy))pentyloxy)phenyl)propanoic Acid);
99mTc(tricine)(TPPTS)(3-(6-(3-N-(2-(2-(2-(2-(2-(2-(2-(2-((6-(diazenido)(3-pyridyl))carbonylamino)ethoxy)-ethoxy)ethoxy)ethoxy)ethoxy) ethoxy)ethoxy)ethyl)carbamoyl)propoxy)-2-(5-(5-oxo-1-prop-2-enyl(2-6,7,8-trihydronaphthyloxy))pentyloxy)phenyl)propanoic Acid);
99mTc(tricine)(TPPTS)(3-(6-(3-N-(5-((6-(diazenido)(3-pyridyl))carbonylamino)-5-(N-(2,3,4,5,6-pentahydroxyhexyl)carbamoyl)pentyl)carbamoyl)propoxy-2-(5-(5-oxo-1-prop-2-enyl(2-6,7,8-trihydronaphthyloxy))pentyloxy)phenyl)propanoic Acid);
99mTc(tricine)(TPPTS)(3-(6-(3-N-(5-((6-(diazenido)(3-pyridyl))carbonylamino)-5-(N-(6-deoxy-p-cyclodextryl)carbamoyl)pentyl)carbamoyl)propoxy-2-(5-(5-oxo-1-prop-2-enyl(2-6,7,8-trihydronaphthyloxy))pentyloxy)phenyl)propanoic Acid);
99mTc(tricine)(TPPTS)(3-(6-(3-(N-(3-((6-((diazenido)(3-pyridyl))-Gly-Lys-Lys-Lys)aminopropyl)carbamoyl)propoxy)-2-(5-(5-oxo-1-prop-2-enyl(2-6,7,8-trihydronaphthyloxy))pentyloxy)phenyl)propanoic Acid);
99mTc(tricine)(TPPTS)((E)-N-[3-(6-diazenidonicotinamido)propyl]-3-[6-[[(2,6-dichlorophenyl)thio]methyl]-3-(2-phenylethoxy)-2-pyridinyl]-2-propenamide);
99mTc(tricine) (TPPTS)((E)-N-[3-(6-diazenidonicotinamido)propyl]-3-[6-[(phenylthio)methyl]-3-(2-phenylethoxy)-2-pyridinyl]-2-propenamide);
99mTc(tricine)(TPPTS)((E)-N-[3-(6-diazenidonicotinamido)propyl]-3-[6-[[(2-chlorophenyl)thio]methyl]-3-(2-phenylethoxy)-2-pyridinyl]-2-propenamide);
99mTc(tricine)(TPPTS)((E)-N-[3-(6-diazenidonicotinamido)propyl]-3-[6-[[(2,6-dimethylphenyl)thio]methyl]-3-(2-phenylethoxy)-2-pyridinyl]-2-propenamide);
99mTc(tricine)(TPPTS)((E)-N-[3-(6-diazenidonicotinamido)propyl]-3-[6-[[(2,3,5,6-tetrafluorophenyl)thio]methyl]-3-(2-phenylethoxy)-2-pyridinyl]-2-propenamide);
99mTc(tricine) (TPPTS)((E)-N-[3-(6-diazenidonicotinamido)propyl]-3-[6-[[(2,3,5,6-tetrafluorophenyl)thio]methyl]-3-(2-phenylethoxy)-2-pyridinyl]-2-propenamide);
99mTc(tricine) (TPPTS)((E)-N-[2-(6-diazenidonicotinamido)ethyl]-3-[6-[[(2,6-dichlorophenyl)thio]methyl]-3-(2-phenylethoxy)-2-pyridinyl]-2-propanamide);
99mTc(tricine)(TPPTS)(6-[6-(6-diazenidonicotinamido)-4,4-dimethylpentyloxy]-5-(2-propenyl)-1,2,3,4-tetrahydronaphthalen-1-one);
99mTc(tricine)(TPPTS)(2-[[[5-[[2,2-Dimethyl-6-[(4-(3,4-methylenedioxyphenyl)-6-phenyl-2-pyridinyl)oxy]-1-hexanamino]carbonyl]-2-pyridinyl]diazenido);
99mTc(tricine)(TPPTS)(2-[[[5-[[N-[6-[(4,6-diphenyl-2-pyridinyl)oxy]-hexanoyl]-glycine-alpha-amino]carbonyl]-2-pyridinyl]diazenido);
99mTc(tricine)(TPPTS)(2,4-Diethyl-[5-[5,5-dimethyl-6-[[6-[[diazenido]-3-pyridinyl]carbonyl]amino]hexyl]oxy]phenol);
99mTc(tricine)(TPPTS)(2-((6-(diazenido)(3-pyridyl)carbonylamino)-3-(2-(5-(4,6-diphenyl(2-pyridyloxy))pentyloxy)phenyl)propanoic acid);
99mTc(tricine)(TPPTS)(3-((6-(diazenido)(3-pyridyl)carbonylamino)-3-(N-(6-(4,6-diphenyl(2-pyridyloxy))-2,2-dimethylhexyl)carbamoyl)propanoic acid);
99mTc(tricine)(TPPTS)(2-(2-((6-(diazenido)(3-pyridyl)carbonylamino)-3-carboxypropanoylamino)-3-(2-(5-(4,6-diphenyl(2-pyridyloxy))pentyloxy)phenyl)propanoic acid);
99mTc(tricine)(TPPTS)(2-(2-((5-(N-(2-(N-(3-(2-(2-(3-(2-(2,5-dioxoimidazolidin-4-yl)acetylamino)-propoxy)ethoxy)ethoxy)-propyl)carbamoyl)-1-(N-(6-(4,6-diphenyl(2-pyridyloxy))-2,2-dimethylhexyl)carbamoyl)-ethyl)carbamoyl(2-pyridyl))diazenido);
99mTc(tricine)(TPPTS)(1-(3-((6-(diazenido)-(3-pyridyl)carbonylamino)-3-(N-(6-(4,6-diphenyl(2-pyridyloxy))-2,2-dimethylhexyl)carbamoyl)propanoylamino)-ethane-1,2-dicarboxylic acid);
99mTc(tricine)(TPPTS)(2-(2-((5-(N-(1-(N-(6-(4,6-diphenyl(2-pyridyloxy))-2,2-dimethylhexyl)carbamoyl)-2-(3-(((4,5,6-trihydroxy-3-(hydroxymethyl)(2-oxanyl))amino)carbonylamino)-propanoylamino)ethyl)carbamoyl(2-pyridyl))diazenido);
99mTc(tricine)(TPPTS)(2-(2-((5-((6-(4-benzo[d] 1,3-dioxolan-5-yl-6-phenyl(2-pyridyloxy))-2,2-dimethylhexanoyl-amino)sulfonyl)-(2-pyridyl))diazenido);
99mTc(tricine)(TPPTS)(3-((6-((diazenido)(3-pyridyl)carbonylamino)-3-(N-(6-(4-benzo[d] 1,3-dioxolan-5-yl-6-phenyl(2-pyridyloxy))-2,2-dimethylhexyl)carbamoyl)propanoic acid);
99mTc(tricine)(TFP)(3-((6-((diazenido)(3-pyridyl)carbonylamino)-3-(N-(6-(4-benzo[d] 1,3-dioxolan-5-yl-6-phenyl(2-pyridyloxy))-2,2-dimethylhexyl)carbamoyl)propanoic acid);
99mTc(tricine)(TPPTS)(2-(2-((5-(N-(1-(N-(6-(4-benzo[d] 1, 3-dioxolan-5-yl-6-phenyl(2-pyridyloxy))-2,2-dimethyl-hexyl)carbamoyl)-2-(4-hydroxyphenyl)ethyl)carbamoyl(2-pyridyl))diazenido);
99mTc(tricine)(TPPTS)(2-((6-(diazenido)(3-pyridyl)carbonylamino)-2-(6-(4-benzo[d] 1,3-dioxolan-5-yl-6-phenyl(2-pyridyloxy))-2,2-dimethylhexanoylamino)acetic acid);
99mTc(tricine)(TPPTS)(2-(2-((5-(N-(5-((3-((N-(6-(4,6-diphenyl(2-pyridyloxy))-2,2-dimethylhexanoylamino)-3-(4-hydroxyphenyl)propanoylamino)-1-(N-(2,3,4,5,6-pentahydroxyhexyl)carbamoyl)pentyl)carbamoyl(2-pyridyl))diazenido);
99mTc(tricine)(TPPTS)(2-(2-((5-(N-(5-((3-((N-(6-(4-benzo[d] 1,3-dioxolan-5-yl-6-phenyl(2-pyridyloxy))-2,2-dimethylhexyl)-carbamoyl)-2-(N-(2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-ethyl)carbamoyl(2-pyridyl))diazenido);
99mTc(tricine)(TPPTS)(2-(2-((5-(N-(5-((3-((N-(6-(4-benzo[d] 1,3-dioxolan-5-yl-6-phenyl(2-pyridyloxy))-2,2-dimethylhexyl)carbamoyl)amino)phenyl)carbonylamino)-1-(N-(2,3,4,5,6-pentahydroxyhexyl)carbamoyl)pentyl)carbamoyl(2-pyridyl))diazenido);
99mTc(tricine)(TPPTS)(2-((6-(diazenido)(3-pyridyl))carbonylamino)-3-(N-(6-(4-benzo[d] 1,3-dioxolan-5-yl-6-phenyl(2-pyridyloxy))-2,2-dimethylhexyl)carbamoyl)-propanoylamino)-3-carboxypropanoylamino)-3-carboxypropanoylamino)-ethane-1,2-dicarboxylic acid);
99mTc(tricine)(TPPTS)(2-((6-(diazenido)(3-pyridyl))carbonylamino)-3-(2-(5-(4-benzo[d] 1,3-dioxolan-5-yl-6-phenyl(2-pyridyloxy))pentyloxy)phenyl)propanoic acid);
99mTcO(4-ethyl-2-(4-fluorophenyl)-5-[(5,5-dimethyl-6-aminohexyl)oxy]phenol N-[4-(carboxy)benzyl]-N,Nxe2x80x2-bis[2-thiolatoethyl]-glycinamide);
99mTcO(N-[2,2-Dimethyl-6-[(4-(3,4-methylenedioxyphenyl)-6-phenyl-2-pyridinyl)oxy]-hexyl]-bis(mercapto-acetyl)pentanoate); and
111In(2-({2-[((2S)-2-[Bis(carboxymethyl)amino]-3-{4-[({[3-(2-{2-[3-(5-{5-[5-(4,6-diphenyl(2-pyridyloxy))-1,1-dimethylpentyl](5H-1,2,3,4-tetraazolyl)}pentanoylamino)-propoxy]ethoxy}ethoxy)propyl]amino}thioxomethyl)amino]phenyl}propyl)(carboxymethyl)amino]ethyl}-(carboxymethyl)amino)acetate).
[27] In an embodiment, the present invention provides a kit comprising a reagent of any one of Embodiment [22-26] and a perfusion imaging agent.
[28] In an embodiment, the present invention provides a kit of Embodiment [27] further comprising a reducing agent.
[29] In an embodiment, the present invention provides a kit of Embodiment [28] wherein the reducing agent is tin(II).
[30] In an embodiment, the present invention provides a kit of Embodiment [29] further comprising one or more ancillary ligands.
[31] In an embodiment, the present invention provides a kit of Embodiment [30] wherein the ancillary ligands are tricine and TPPTS.
[32] In an embodiment, the present invention provides a kit comprising radiolabeled LTB4 binding agent of Embodiment [18] and a perfusion imaging agent.
[33] In an embodiment, the present invention provides a kit of Embodiment [32] further comprising a reducing agent.
[34] In an embodiment, the present invention provides a kit of Embodiment [33] wherein the reducing agent is tin(II).
[35] In an embodiment, the present invention provides a kit of Embodiment [34] further comprising one or more ancillary ligands.
[36] In an embodiment, the present invention provides a kit of Embodiment [35] wherein the ancillary ligands are tricine and TPPTS.
[37] In an embodiment, the present invention provides a kit comprising radiolabeled LTB4 binding agent of Embodiment [18] and a radiolabeled perfusion imaging agent of any one of Embodiments [22-26].
[38] In an embodiment, the present invention provides a kit of Embodiment [37] further comprising a reducing agent.
[39] In an embodiment, the present invention provides a kit of Embodiment [38] wherein the reducing agent is tin(II).
[40] In an embodiment, the present invention provides a kit of Embodiment [39] further comprising one or more ancillary ligands.
[41] In an embodiment, the present invention provides a kit of Embodiment [40] wherein the ancillary ligands are tricine and TPPTS.
[42] In an embodiment, the present invention provides a method according to embodiment [1] further comprising forming an image from the detection of said agents.
[43] In an embodiment, the present invention provides a method according to embodiment [42] wherein the images are displayed side-by-side to faciliate interpretation of the localization of the radiolabeled LTB4 binding agent in the body, relative to the distribution of the radiolabeled perfusion agent in the body.
[44] In an embodiment, the present invention provides a method according to embodiment [42] wherein the images are are overlayed to faciliate interpretation of the localization of the radiolabeled LTB4 binding agent in the body, relative to the distribution of the radiolabeled perfusion agent in the body.
[45] In an embodiment, the present invention provides a method of diagnosing and localizing sites of inflammation and perfusion abnormalities in a single dual isotope imaging procedure.
[46] In an embodiment, the present invention provides a method of diagnostic imaging comprising the concurrent detection and localization of sites of ischemic tissue injury and perfusion abnormalities in a single imaging procedure.
[47] In an embodiment, the present invention provides a method according to embodiment [46] wherein the imaging procedure is one or more procedure selected from the group, CT imaging, ultrasound, and scintigraphy.
[48] In an embodiment, the present invention provides a method of diagnosing and localizing sites of inflammation and perfusion abnormalities in a single dual imaging procedure.
[49] In an embodiment, the present invention provides a method according to embodiment [48] wherein the imaging procedure is one or more procedure selected from the group, CT imaging, ultrasound, and scintigraphy.
[50] In an embodiment, the present invention provides a method of diagnostic imaging comprising the concurrent detection and localization of sites of of ischemic tissue injury (e.g. reperfusion injury), vulnerable plaque, bacterial endocarditis or cardiac transplant rejection in a single dual imaging procedure.
[51] In an embodiment, the present invention provides a method according to embodiment [50] wherein the imaging procedure is one or more procedure selected from the group, CT imaging, ultrasound, and scintigraphy.
[52] In an embodiment, the present invention provides a method of diagnostic imaging comprising the concurrent detection and localization of sites of vulnerable plaque and perfusion abnormalities in a single imaging procedure.
[53] In an embodiment, the present invention provides a method according to embodiment [52] wherein the imaging procedure is one or more procedure selected from the group, CT imaging, ultrasound, and scintigraphy.
[54] In an embodiment, the present invention provides a method of diagnostic imaging comprising the concurrent detection and localization of sites of cardiac infection and perfusion abnormalities in a single imaging procedure.
[55] In an embodiment, the present invention provides a method according to embodiment [54] wherein the imaging procedure is one or more procedure selected from the group, CT imaging, ultrasound, and scintigraphy.
[56] In an embodiment, the present invention provides a method of diagnostic imaging comprising the concurrent detection and localization of sites of cardiac transplant rejection and perfusion abnormalities in a single imaging procedure.
[57] In an embodiment, the present invention provides a method according to embodiment [56] wherein the imaging procedure is one or more procedure selected from the group, CT imaging, ultrasound, and scintigraphy.
[58] In an embodiment, the present invention provides a method according to embodiment [1] wherein the radiolabeled LTB4 binding agent is a reagent capable of direct transformation into a compound radiolabeled with a radioisotope selected from the group consisting of, 123I, 125I, 18F, 11C, 13N, 150O, and 75Br, and said reagent having the formula:
Wexe2x80x94Xxe2x80x94Lnxe2x80x94Y 
wherein,
We is selected from the group: 
xe2x80x83wherein,
A1 is N, Cxe2x80x94OH, or CH;
A2 and A3 are independently N or CH;
A4 is N or CR3;
A5 is 0 or S;
A6 is 0, CH2 or S;
A7 is Cxe2x80x94OH, N, NH, 0 or S;
A8 is NH, CH2, O, S, N, or CH;
A9 is N or CH;
a and b indicate the alternative positions of a double bond;
R1 is selected from the group: H, xe2x80x94C(xe2x95x90NH)NH2, C1-C6 alkyl substituted with 0-3 R4, C1-C6 alkoxy substituted with 0-3 R4, aryl substituted with 0-3 R5, and heterocycle substituted with 0-3 R5;
R2 is selected from the group: H, C1-C3 alkyl, C2-C3 alkenyl, cyclopropyl, cyclopropylmethyl, and aryl substituted with 0-3 R5;
R3 is xe2x80x94H, xe2x80x94OH or C1-C3 alkoxy; or alternatively, R1 and R3 can be taken together with the atoms to which they are attached to form a fused phenyl ring substituted with 0-3 R5;
R4 is independently selected from the group: xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x95x90O, xe2x80x94N(R6)(R7), and xe2x80x94CF3;
R5 is independently selected from the group: xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94N(R6)(R7), xe2x80x94CF3, C1-C3 alkyl, C1-C3 alkoxy, and methylenedioxy;
R6 and R7 are independently H or C1-C3 alkyl; provided that when A1 and A2 are CH, A3 is Cxe2x80x94X, and A4 is CR3, R1 is selected from the group: C1-C5 alkyl substituted with 1-3 R4, C1-C5 alkoxy substituted with 0-3 R4, and aryl substituted with 0-3 R5;
X is O, S, CH2 or CHxe2x95x90CH;
Ln is a linking group having the formula
(CR8R9)g(W1)hxe2x80x94(M1)kxe2x80x94(CR10R11)g, 
xe2x80x83wherein,
R8, R9, R10 and R11 are independently selected at each occurrence from the group: H, C1-C5 alkyl, and C1-C5 alkoxy, or alternatively, R8 and R9 or R10 and R11 may be taken together to form a 3-6 membered cycloalkyl or heterocycle;
W1 is independently selected from the group: O, S, C(xe2x95x90O)O, OC(xe2x95x90O), CHxe2x95x90CH, (OCH2CH2)p and (CH2CH2O)pxe2x80x2, wherein p and pxe2x80x2 are independently 1-3;
M1 is selected from the group: phenyl substituted with 0-3 R12, heterocycle substituted with 0-3 R12, benzophenone substituted with 0-3 R12, and diphenylether substituted with 0-3 R12;
R12 is independently selected from the group: xe2x80x94COOR13, C1-C5 alkyl substituted with 0-3 R14, and C1-C5 alkoxy substituted with 0-3 R14;
R13 is H or C1-C5 alkyl:
R14 is xe2x80x94COOH;
g is 0-10;
h is 0-3;
k is 0-1;
gxe2x80x2 is 0-5;
provided that when h is 0 and k is 0, g is  greater than 1;
and provided that when W1 is O or S and k is 0, g+gxe2x80x2 is xe2x89xa71; and
Y is selected from C(xe2x80x94O)NH, NHC(xe2x80x94O), Cxe2x95x90O, C(xe2x80x94O)O, OC(xe2x95x90O), NHS(xe2x95x90O)2, C(xe2x95x90O)NHS(xe2x95x90O)2, COOH, C(xe2x95x90O)NH2, NH(Cxe2x95x90O)NH, or tetrazole; or
a pharmaceutically acceptable salts thereof.
[59] In an embodiment, the present invention provides a method according to embodiment [58] wherein:
we is selected from the group: 
xe2x80x83wherein,
A1 is N, Cxe2x80x94OH, or CH;
A2 and A3 are CH;
A4 is CR3;
A5 is O;
A6 is O or CH2;
R4 is independently selected from the group:
A5 is O;
A6 is O or CH2;
R4 is independently selected from the group: xe2x80x94F, xe2x80x94Cl, xe2x95x90O, xe2x80x94N(R6)(R7), and xe2x80x94CF3;
R5 is independently selected from the group: xe2x80x94F, xe2x80x94Cl, xe2x80x94CF3, C1-C3 alkyl, C1-C3 alkoxy, and methylenedioxy;
X is O, CH2 or CHxe2x95x90CH; and
R8, R9, R10 and R11 are independently selected at each occurrence from the group: H, C1-C5 alkyl, and C1-C5 alkoxy;
or alternatively, R8 and R9 or R10 and R11 may be taken together to form a 3-6 membered cycloalkyl.
[60] In an embodiment, the present invention provides a reagent of Embodiment [58] wherein:
R1 is selected from the group:
H, xe2x80x94C(xe2x95x90NH)NH2, C1-C6 alkyl substituted with 0-2 R4, C1-C6 alkoxy substituted with 0-2 R4, aryl substituted with 0-2 R5, and heterocycle substituted with 0-2 R5;
R3 is xe2x80x94H, xe2x80x94OH or C1-C3 alkoxy;
or alternatively, R1 and R3 can be taken together with the atoms to which they are attached to form a fused phenyl ring substituted with 0-2 R5;
R4 is independently selected from the group: xe2x95x90O, and xe2x80x94N(R6)(R7);
R5 is independently selected from the group: xe2x80x94F, C1-C3 alkyl, C1-C3 alkoxy, and methylenedioxy;
X is O, CH2 or CHxe2x95x90CH;
W1 is O;
M1 is selected from the group: phenyl substituted with 0-1 R12, heterocycle substituted with 0-1 R12, benzophenone substituted with 0-1 R12, and diphenylether substituted with 0-1 R12;
R12 is independently selected from the group: xe2x80x94COOR13, C1-C5 alkyl substituted with 0-1 R14, and C1-C5 alkoxy substituted with 0-1 R14; and
M2 is selected from the group: aryl substituted with 0-1 R19, cycloalkyl substituted with 0-3 R19, and heterocycle substituted with 0-1 R19.
[61] In an embodiment, the present invention provides a kit comprising a reagent of Embodiment [58] and a perfusion imaging agent.
In a further embodiment, the present invention provides a novel reagent capable of direct transformation into a radiopharmaceutical having a binding affinity for the LTB4 receptor of less than 1000 nM.
In a further embodiment, the present invention provides a novel method of detecting sites of infection and inflammation in a mammal comprising administering to said mammal a radiolabeled LTB4 binding agent and then detecting said sites using a radiation detecting probe.
In a further embodiment, the present invention provides a novel method of imaging sites of infection and inflammation in a mammal comprising administering to said mammal a radiolabeled LTB4 binding agent and then imaging said sites using a planar or ring gamma camera.
In a further embodiment, the present invention provides a novel method of diagnosing disease in a mammal associated with infection and inflamation comprising imaging said mammal using a radiolabeled LTB4 binding agent and determining the presence of said disease.
In a further embodiment, the present invention provides a novel method of dual isotope imaging utilizing an organ perfusion imaging agent and an inflammation image utilizing the LTB4-targeted radiopharmaceutical reagents described herein. The simultaneous imaging of organ perfusion and inflammation allows a more complete assessment of the underlying disease, both in terms of blood flow alterations and inflammatory changes, in a single imaging session on a patient. This simultaneous imaging procedure is more time-efficient than the serial/sequential imaging of perfusion and inflammation which is acquired in two separate imaging sessions. The simultaneous imaging process will save labor time of radiologists and imaging technologists, as well as reduce the time that imaging instruments are utilized in acquiring the perfusion and inflammation images. This procedure will also result in greater patient comfort by reducing the time needed for the subject to remain immobile relative to the situation of two separate imaging procedures. In addition, the simultaneous acquisition process will allow for improved accuracy in the spatial registration (i.e. overlay) of of two separate imaging procedures. In addition, the simultaneous acquisition process will allow for improved accuracy in the spatial registration (i.e. overlay) of perfusion and inflammation images in a patient versus the situation of serial images being co-registered expost-facto. This will allow a better spatial correlation of perfusion abnormalities and inflammatory processes in the tissue (e.g. in comparing areas of perfusion deficits in the heart to the location of inflammatory lesions associated with atherosclerotic plaques of the coronary arteries, and the like).
In a further embodiment, the invention provides a method of diagnostic imaging comprising the concurrent detection of inflammation and organ perfusion in a single imaging procedure. More particularly, a method of diagnostic imaging comprising the concurrent detection and localization of perfusion imaging agent and a LTB4 binding agent conjugated to an imageable moiety, such as a gamma ray or positron emitting radioisotope, a magnetic resonance imaging moiety, an X-ray imaging moiety, or an ultrasound imaging moiety.
An imaging moiety comprising a diagnostically radionuclide (i.e., a radioactive metal ion that has imageable gamma ray or positron emissions) are useful as radioimaging contrast agents.
An imaging moieties comprising a paramagnetic metal ion are useful as magnetic resonance imaging contrast agents An imaging moiety comprising one or more X-ray and a surfactant microsphere, are useful as ultrasound contrast agents.
In another preferred embodiment wherein the LTB4 binding agent conjugated to an X-ray imaging moiety, the metal is selected from the group: Re, Sm, Ho, Lu, Pm, Y, Bi, Pd, Gd, La, Au, Au, Yb, Dy, Cu, Rh, Ag, and Ir.
The frequently used heavy atom in X-ray imaging moieties is iodine. Recently, X-ray contrast agents comprised of metal chelates (Wallace, R., U.S. Pat. No. 5,417,959) and polychelates comprised of a plurality of metal ions (Love, D., U.S. Pat. No. 5,679,810) have been disclosed. More recently, multinuclear cluster complexes have been disclosed as X-ray contrast agents (U.S. Pat. No. 5,804,161, PCT WO91/14460, and PCT WO 92/17215).
X-ray contrast imaging moieties of the present invention are comprised of one or more LTB4 binding agents attached to one or more X-ray absorbing or xe2x80x9cheavyxe2x80x9d atoms of atomic number 20 or greater. The frequently used heavy atom in X-ray contrast agents is iodine. Recently, X-ray contrast agents comprised of metal chelates (Wallace, R., U.S. Pat. No. 5,417,959) and polychelates comprised of a plurality of metal ions (Love, D., U.S. Pat. No. 5,679,810) have been disclosed. More recently, multinuclear cluster complexes have been disclosed as X-ray contrast agents (U.S. Pat. No. 5,804,161, PCT WO91/14460, and PCT WO 92/17215).
In another preferred embodiment wherein the LTB4 binding agent conjugated to a MRI imaging moiety of the present invention are comprised of one or more LTB4 binding agents attached to one or more paramagnetic metal ions. The paramagnetic metal ions are present in the form of metal complexes or metal oxide particles. U.S. Pat. Nos. 5,412,148, and 5,760,191, describes examples of chelators for paramagnetic metal ions for use in MRI contrast agents. U.S. Pat. Nos. 5,801,228, 5,567,411, and 5,281,704, describe examples of polychelants useful for complexing more than one paramagnetic metal ion for use in MRI contrast agents. U.S. Pat. No. 5,520,904, describes particulate compositions comprised of paramagnetic metal ions for use as MRI contrast agents.
In another preferred embodiment wherein the LTB4 binding agent conjugated to an ultrasound contrast imaging moiety of the present invention comprise a plurality of LTB4 binding agents attached to or incorporated into a microbubble of a biocompatible gas, a liquid carrier, and a surfactant microsphere. In this context, the term liquid carrier means aqueous solution and the term surfactant means any amphiphilic material which produces a reduction in interfacial tension in a solution. A list of suitable surfactants for forming surfactant microspheres is disclosed in EP0727225A2, herein incorporated by reference. The term surfactant microsphere includes nanospheres, liposomes, vesicles and the like. The biocompatible gas can be air, or a fluorocarbon, such as a C3-C5 perfluoroalkane, which provides the difference in echogenicity and thus the contrast in ultrasound imaging. The gas is encapsulated or contained in the microsphere to which is attached the LTB4 binding agent, optionally via a linking group. The attachment can be covalent, ionic or by van der Waals forces. Specific examples of such contrast imaging moieties include lipid encapsulated perfluorocarbons with a plurality of tumor neovasculature receptor binding peptides, polypeptides or peptidomimetics.
When any variable occurs more than one time in any constituent or in any formula, its definition on each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-2 R52, then said group may optionally be substituted with up to two R52, and R52 at each occurrence is selected independently from the defined list of possible R52. Also, by way of example, for the group xe2x80x94N(R53)2, each of the two R53 substituents on N is independently selected from the defined list of possible R53. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.
By xe2x80x9creagentxe2x80x9d is meant a compound of this invention capable of direct transformation into a radiopharmaceutical of this invention. Reagents may utilized directly for the preparation of the radiopharmaceuticals of this invention or may be a component in a kit of this invention.
The term xe2x80x9cbinding agentxe2x80x9d means a radiopharmaceutical of this invention having affinity for and capable of binding to LTB4. The binding agents of this invention have Ki less than 1000 nM.
By xe2x80x9cstable compoundxe2x80x9d or xe2x80x9cstable structurexe2x80x9d is meant herein a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious diagnostic agent.
The term xe2x80x9csubstitutedxe2x80x9d, as used herein, means that one or more hydrogens on the designated atom or group is replaced with a selection from the indicated group, provided that the designated atom""s or group""s normal valency is not exceeded, and that the substitution results in a stable compound. When a substituent is keto (i.e., xe2x95x90O), then 2 hydrogens on the atom are replaced.
The term xe2x80x9cbondxe2x80x9d, as used herein, means either a single or double bond.
The term xe2x80x9csaltxe2x80x9d, as used herein, is used as defined in the CRC Handbook of Chemistry and Physics, 65th Edition, CRC Press, Boca Raton, Fla., 1984, as any substance which yields ions, other than hydrogen or hydroxyl ions.
As used herein, xe2x80x9calkylxe2x80x9d is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms; xe2x80x9ccycloalkylxe2x80x9d or xe2x80x9ccarbocyclexe2x80x9d is intended to include saturated and partially unsaturated ring groups, including mono-, bi- or poly-cyclic ring systems, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and adamantyl; xe2x80x9cbicycloalkylxe2x80x9d is intended to include saturated bicyclic ring groups such as [3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane (decalin), [2.2.2]bicyclooctane, and so forth.
As used herein, the term xe2x80x9calkenexe2x80x9d or xe2x80x9calkenylxe2x80x9d is intended to include both branched and straight-chain groups of the formula CnH2nxe2x88x921 having the specified number of carbon atoms.
As used herein, the term xe2x80x9calkynexe2x80x9d or xe2x80x9calkynylxe2x80x9d is intended to include both branched and straight-chain groups of the formula CnH2nxe2x88x923 having the specified number of carbon atoms.
As used herein, xe2x80x9carylxe2x80x9d or xe2x80x9caromatic residuexe2x80x9d is intended to mean phenyl or naphthyl, which when substituted, the substitution can be at any position.
As used herein, the term xe2x80x9cheterocyclexe2x80x9d or xe2x80x9cheterocyclic ring systemxe2x80x9d is intended to mean a stable 5- to 7-membered monocyclic or bicyclic or 7- to 14-membered bicyclic or tricyclic heterocyclic ring which may be saturated, partially unsaturated, or aromatic, and which consists of carbon atoms and from 1 to 4 heteroatoms selected independently from the group consisting of N, O and S and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen may optionally be quaternized, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. The heterocyclic ring may be attached to its pendant group at any heteroatom or carbon atom which results in a stable structure. The heterocyclic rings described herein may be substituted on carbon or on a nitrogen atom if the resulting compound is stable. Examples of such heterocycles include, but are not limited to, benzopyranyl, thiadiazine, tetrazolyl, benzofuranyl, benzothiophenyl, indolene, quinoline, isoquinolinyl or benzimidazolyl, piperidinyl, 4-piperidone, 2-pyrrolidone, tetrahydrofuran, tetrahydroquinoline, tetrahydroisoquinoline, decahydroquinoline, octahydroisoquinoline, azocine, triazine (including 1,2,3-, 1,2,4-, and 1,3,5-triazine), 6H-1,2,5-thiadiazine, 2H,6H-1,5,2-dithiazine, thiophene, tetrahydrothiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, xanthone, phenoxathiin, 2H-pyrrole, pyrrole, imidazole, pyrazole, thiazole, isothiazole, oxazole (including 1,2,4- and 1,3,4-oxazole), isoxazole, triazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, 3H-indole, indole, 1H-indazole, purine, 4H-quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, 4aH-carbazole, carbazole, xcex2-carboline, phenanthridine, acridine, perimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, isochroman, chroman, chromanone, pyrrolidine, pyrroline, imidazolidine, imidazoline, pyrazolidine, pyrazoline, piperazine, indoline, isoindoline, quinuclidine, or morpholine. Also included are fused ring and spiro compounds containing, for example, the above heterocycles.
As used herein, the term xe2x80x9calkarylxe2x80x9d means an aryl group bearing an alkyl group of 1-10 carbon atoms; the term xe2x80x9caralkylxe2x80x9d means an alkyl group of 1-10 carbon atoms bearing an aryl group; the term xe2x80x9carylalkarylxe2x80x9d means an aryl group bearing an alkyl group of 1-10 carbon atoms bearing an aryl group; and the term xe2x80x9cheterocycloalkylxe2x80x9d means an alkyl group of 1-10 carbon atoms bearing a heterocycle.
A xe2x80x9cpolyalkylene glycolxe2x80x9d is a polyethylene glycol, polypropylene glycol or polybutylene glycol having a molecular weight of less than about 5000, terminating in either a hydroxy or alkyl ether moiety.
A xe2x80x9ccarbohydratexe2x80x9d is a polyhydroxy aldehyde, ketone, alcohol or acid, or derivatives thereof, including polymers thereof having polymeric linkages of the acetal type.
A xe2x80x9ccyclodextrinxe2x80x9d is a cyclic oligosaccharide. Examples of cyclodextrins include, but are not limited to, xcex1-cyclodextrin, hydroxyethyl-xcex1-cyclodextrin, hydroxypropyl-xcex1-cyclodextrin, xcex2-cyclodextrin, hydroxypropyl-xcex2-cyclodextrin, carboxymethyl-xcex2-cyclodextrin, dihydroxypropyl-xcex2-cyclodextrin, hydroxyethyl-xcex2-cyclodextrin, 2,6 di-O-methyl-xcex2-cyclodextrin, sulfated-xcex2-cyclodextrin, xcex3-cyclodextrin, hydroxypropyl-xcex3-cyclodextrin, dihydroxypropyl-xcex3-cyclodextrin, hydroxyethyl-xcex3-cyclodextrin, and sulfated xcex3-cyclodextrin.
As used herein, the term xe2x80x9cpolycarboxyalkylxe2x80x9d means an alkyl group having between two and about 100 carbon atoms and a plurality of carboxyl substituents; and the term xe2x80x9cpolyazaalkylxe2x80x9d means a linear or branched alkyl group having between two and about 100 carbon atoms, interrupted by or substituted with a plurality of amine groups.
A xe2x80x9creducing agentxe2x80x9d is a compound that reacts with the radionuclide, which is typically obtained as a relatively unreactive, high oxidation state compound, to lower its oxidation state by transfering electron(s) to the radionuclide, thereby making it more reactive. Reducing agents useful in the preparation of radiopharmaceuticals and in diagnostic kits useful for the preparation of said radiopharmaceuticals include but are not limited to stannous chloride, stannous fluoride, formamidine sulfinic acid, ascorbic acid, cysteine, phosphines, and cuprous or ferrous salts. Other reducing agents are described in Brodack et. al., PCT Application 94/22496, which is incorporated herein by reference.
A xe2x80x9ctransfer ligandxe2x80x9d is a ligand that forms an intermediate complex with the radionuclide that is stable enough to prevent unwanted side-reactions but labile enough to be converted to the radiopharmaceutical. The formation of the intermediate complex is kinetically favored while the formation of the radiopharmaceutical is thermodynamically favored. Transfer ligands useful in the preparation of radiopharmaceuticals and in diagnostic kits useful for the preparation of said radiopharmaceuticals include but are not limited to gluconate, glucoheptonate, mannitol, glucarate, N,N,Nxe2x80x2,Nxe2x80x2-ethylenediaminetetraacetic acid, pyrophosphate and methylenediphosphonate. In general, transfer ligands are comprised of oxygen or nitrogen donor atoms.
The term xe2x80x9cdonor atomxe2x80x9d refers to the atom directly attached to a metal by a chemical bond.
xe2x80x9cAncillaryxe2x80x9d or xe2x80x9cco-ligandsxe2x80x9d are ligands that are incorporated into the radiopharmaceutical during its synthesis. They serve to complete the coordination sphere of the radionuclide together with the chelator or radionuclide bonding unit of the reagent. For radiopharmaceuticals comprised of a binary ligand system, the radionuclide coordination sphere is composed of one or more chelators or bonding units from one or more reagents and one or more ancillary or co-ligands, provided that there are a total of two types of ligands, chelators or bonding units. For example, a radiopharmaceutical comprised of one chelator or bonding unit from one reagent and two of the same ancillary or co-ligands and a radiopharmaceutical comprised of two chelators or bonding units from one or two reagents and one ancillary or co-ligand are both considered to be comprised of binary ligand systems. For radiopharmaceuticals comprised of a ternary ligand system, the radionuclide coordination sphere is composed of one or more chelators or bonding units from one or more reagents and one or more of two different types of ancillary or co-ligands, provided that there are a total of three types of ligands, chelators or bonding units. For example, a radiopharmaceutical comprised of one chelator or bonding unit from one reagent and two different ancillary or co-ligands is considered to be comprised of a ternary ligand system.
Ancillary or co-ligands useful in the preparation of radiopharmaceuticals and in diagnostic kits useful for the preparation of said radiopharmaceuticals are comprised of one or more oxygen, nitrogen, carbon, sulfur, phosphorus, arsenic, selenium, and tellurium donor atoms. A ligand can be a transfer ligand in the synthesis of a radiopharmaceutical and also serve as an ancillary or co-ligand in another radiopharmaceutical. Whether a ligand is termed a transfer or ancillary or co-ligand depends on whether the ligand remains in the radionuclide coordination sphere in the radiopharmaceutical, which is determined by the coordination chemistry of the radionuclide and the chelator or bonding unit of the reagent or reagents.
A xe2x80x9cchelatorxe2x80x9d or xe2x80x9cbonding unitxe2x80x9d is the moiety or group on a reagent that binds to a metal radionuclide through the formation of chemical bonds with one or more donor atoms.
The term xe2x80x9cbinding sitexe2x80x9d means the site in vivo or in vitro that binds a biologically active molecule.
A xe2x80x9cdiagnostic kitxe2x80x9d or xe2x80x9ckitxe2x80x9d comprises a collection of components, termed the formulation, in one or more vials which are used by the practising end user in a clinical or pharmacy setting to synthesize the radiopharmaceutical. The kit provides all the requisite components to synthesize and use the radiopharmaceutical except those that are commonly available to the practising end user, such as water or saline for injection, a solution of the radionuclide, equipment for heating the kit during the synthesis of the radiopharmaceutical, if required, equipment necessary for administering the radiopharmaceutical to the patient such as syringes and shielding, and imaging equipment.
A xe2x80x9cbufferxe2x80x9d is a compound that is used to control the pH of the kit during its manufacture and during the synthesis of the radiopharmaceutical.
A xe2x80x9clyophilization aidxe2x80x9d is a component that has favorable physical properties for lyophilization, such as the glass transition temperature, and is added to the diagnostic kit to improve the physical properties of the combination of all the components of the kit for lyophilization.
A xe2x80x9cstabilization aidxe2x80x9d is a component that is added to the radiopharmaceutical or to the diagnostic kit either to stabilize the radiopharmaceutical once it is synthesized or to prolong the shelf-life of the kit before it must be used. Stabilization aids can be antioxidants, reducing agents or radical scavengers and can provide improved stability by reacting preferentially with species that degrade other components or the radiopharmaceutical.
A xe2x80x9csolubilization aidxe2x80x9d is a component that improves the solubility of one or more other components in the medium required for the synthesis of the radiopharmaceutical.
A xe2x80x9cbacteriostatxe2x80x9d is a component that inhibits the growth of bacteria in the diagnostic kit either during its storage before use of after the kit is used to synthesize the radiopharmaceutical.
The term xe2x80x9cdual isotope imagingxe2x80x9d means the concurrent scintigraphic imaging of two spectrally-separable gamma emitting (including PET) isotopes wherein one isotope is associated with a LTB4-targeted radiopharmaceutical and the other isotope is associated with an organ perfusion imaging radiopharmaceutical.
The term xe2x80x9cperfusion imaging agentxe2x80x9d means a radiopharmaceutical which distributes within an organ (e.g. heart, brain, kidney) in proportion to the regional blood flow pattern within that organ, allowing for a scintigraphic image to be acquired which represents a picture of relative perfusion of the organ.
The term xe2x80x9csite of endothelial damagexe2x80x9d means a locus of vascular endothelium wherein the endothelial cells have been damaged by mechanical, hemodynamic or biochemical means.
The term xe2x80x9csite of vulnerable plaquexe2x80x9d means a vascular region of active atherosclerosis wherein the endothelium has been damaged and a localized cellular inflammatory process is ongoing.
In one embodiment this invention is a radiolabeled LTB4 antagonist radiopharmaceutical. The radiolabel is a suitable radioisotope having an emission that can be detected outside the body after injection of the radiolabeled LTB4 antagonist into a mammal. Detection using a gamma camera results in an image of the areas of localization of white blood cells bearing the LTB4 receptor to which is attached the radiopharmaceutical. Our approach in designing LTB4 antagonist radiopharmaceuticals was to identify common features in compounds known to have potential therapeutic uses, and then, assisted by a 3-dimensional map of the LTB4 receptor we developed, design radiopharmaceuticals having such features.
A number of therapeutic LTB4 compounds are known. These display a wide variety of structural types. One similarity shared by many of these compounds is the presence of two key regions in the molecule, described in the literature as the eastern and western ends of the molecule, connected by a flexible tethering group. Recent reviews of LTB4 antagonists include Djuric et. al., Drugs of the Future, 1992, 17, pp 819-830; Cohen, N. and Yagaloff, K., Curr. Opin. Invest. Drugs, 1994, 3, pp. 13-22; and Brooks, C. and Summers, J., J. Med. Chem., 1996, 39, pp 2629-2654, the disclosures of which are herein incorporated by reference in their entirety.
We have identified two concepts for designing radiolabeled LTB4 antagonists. In one concept the radioisotope bonding unit is incorporated into the structure in such a way that it participates in the binding of the compound to the receptor site even when bound to the radioisotope. In the second concept, the radioisotope bonding unit is incorporated into a site on the molecule which is not part of the recognition site, and is removed enough from the recognition site that its presence does not interfere with the binding of the compound to the receptor.
An example of the first concept is to design a LTB4 radiopharmaceutical wherein either the eastern or western end of a potential therapeutic LTB4 antagonist is replaced with an appropriate radionuclide bonding unit bound to Tc-99m or a radiohalogen substituent. Scheme 1 shows the potential therapeutic LTB4 antagonist, (I), which has excellent affinity for the LTB4 receptor (Sawyer et al.; J. Med. Chem., 1995, 38, 4411-32). In (I) the tetrazole substituent serves as a hydrogen bonding acceptor, thereby promoting binding of the compound to the receptor. When the tetrazole is absent, the compound has no affinity (7 xcexcM) for the LTB4 receptor. Also shown in Scheme I is radiopharmaceutical (II), which is a LTB4 receptor antagonist labeled with Tc-99m. In this radiopharmaceutical, the tetrazole group of (I) is replaced with the HYNIC metal chelator complexed to Tc, whose coordination sphere is completed by two ancillary ligands. (II) retains good activity for the LTB4 receptor. (II) can be prepared from reagent (Iia), which bears a hydrazone protected hydrazonicotinamide group, by reaction of (Iia) with Tc-99m in the presence of a suitable reducing agent and appropriate ancillary ligands. (Iia) retains very good affinity for LTB4 (Ki=8 nM compared to 3 nM for I). 
An example of the second approach is shown in Scheme 2. Compound III is an active LTB4 antagonist (Ki=41 nm). This compound was elaborated into a reagent of this invention. This was accomplished by conjugation of a hydrazone protected hydrazinonicotinamide group via a three carbon tether to the tyrosine hydroxyl oxygen to provide a reagent for preparing a Tc-99m radiopharmaceutical of the present invention, reagent IV. Reagent (IV), with Ki=52 nM, has essentially the same affinity for LTB4 as does (III). Reagent IV is readily converted to the radiolabeled analog using the methods described below.
The tyrosine aromatic ring of (III) can also be radioiodinated to form a radiopharmaceutical of the present invention. 
In the reagents of the present invention, compounds Iia and IV shown above, the three common structural features are: a western end comprised of a hydrogen bond acceptor, either phenolic oxygen or the pyridine nitrogen, and an aromatic substituent; a spacer or tether; and an eastern end comprised of a hydrogen bond acceptor, a carbonyl oxygen. Some examples of alternative western end moieties are shown in Scheme 3. 
Some examples of alternative spacers or tethers include acyclic alkyl, either straight chain or branched and heterocycloalkyl. Some examples of alternative eastern ends bearing an optional second spacer or tether and a chelator or metal bonding unit are shown in Scheme 4.
The second spacer or tether provide a means of incorporating a pharmacokinetic modifier into the radiopharmaceuticals of the present invention. The pharmacokinetic modifier serves to direct the biodistibution of the portion of the injected radiopharmaceutical that does not become associated with white blood cells. A wide variety of functional groups can serve as pharmacokinetic modifiers, including, but not limited to, carbohydrates, polyalkylene glycols, peptides or other polyamino acids, and cyclodextrins. The modifiers are generally characterized by a plurality of atoms selected from oxygen and nitrogen, which provide enhanced hydrophilicity to the radiopharmaceuticals and can thus affect their rate of blood clearance and the route of elimination. Preferred pharmacokinetic modifiers are those that result in moderate blood clearance and enhanced renal excretion.
Other radiopharmaceuticals of the present invention are comprised of more compact LTB4 antagonist moieties to which are attached an optional spacer or tether and a chelator or metal bonding unit. Examples of these compact LTB4 antagonist moieties are shown in Scheme 5.
The radiolabeled LTB4 antagonist compounds of the present invention can be synthesized using standard synthetic methods known to those skilled in the art, using radioisotopes of halogens (such as chlorine, fluorine, bromine and iodine), technetium and indium, as well as others. Preferable radioisotopes include 123I, 125I, 131I, 99mTc, and 111In.
The LTB4 antagonist compounds of the invention may be labeled either directly (that is, by incorporating the radiolabel directly into the compounds) or indirectly (that is, by incorporating the radiolabel into the compounds through a chelator which has been incorporated into the compounds. For direct labeling, as those skilled in the art will recognize, the labeling may be isotopic or nonisotopic. With isotopic labeling, one group already present in the cyclic compound is substituted with (exchanged for) the radioisotope. With nonisotopic labeling, the radioisotope is added to the cyclic compounds without substituting with (exchanging for) an already existing group.
Generally, labeled compounds are prepared by procedures which introduce the labeled atom at a late stage of the synthesis. This allows for maximum radiochemical yields, and reduces the handling time of radioactive materials. When dealing with short half-life isotopes, a major consideration is the time required to conduct synthetic procedures, and purification methods. Protocols for the synthesis of radiopharmaceuticals are described in Tubis and Wolf, Eds., xe2x80x9cRadiopharmacyxe2x80x9d, Wiley-Interscience, New York (1976); Wolf, Christman, Fowler, Lambrecht, xe2x80x9cSynthesis of Radiopharmaceuticals and Labeled Compounds Using Short-Lived Isotopesxe2x80x9d, in Radiopharmaceuticals and Labeled Compounds, Vol 1, p. 345-381 (1973), the disclosures of each of which are hereby incorporated herein by reference, in their entirety.
Various procedures may be employed in preparing the radiolabeled compounds of the invention where the radiolabel is a halogen. Some common synthetic methodologies for isotopic halogen labeling of aromatic compounds such as the type present here are iododediazonization, iododeborobation, iododestannylation, iododesilation, iododethallation, and halogen exchange reactions. The most common synthetic methodology for nonisotopic halogen labeling of aromatic compounds such as the type present here is iododeprotonation or electrophilic aromatic substitution reactions. These methods and additional procedures are described in Merkushev, Synthesis, 923 (1988), and Seevers et al, Chem. Rev., 82: 575 (1982), the disclosures of each of which are hereby incorporated herein by reference, in their entirety.
Alternatively, such compounds may prepared by way of isotopic labeling from the unlabeled bromo or iodo derivatives by various two step reaction sequences, such as through the use of trialkylsilyl synthons as described in Wilson et at J. Org. Chem., 51: 483 (1986) and Wilbur et al J. Label. Compound. Radiopharm., 19: 1171 (1982), the use of trialkylsilyl synthons as described in Chumpradit et al J. Med. Chem., 34: 877 (1991) and Chumpradit et al J. Med. Chem., 32: 1431 (1989), and the use of boronic acid synthons as described in Kabalka et al J. Label. Compound. Radiopharm., 19: 795 (1982) and Koch et al Chem. Ber., 124:2091 (1991).
The unlabeled iodo compounds are versatile precursors which can be converted to the labeled derivatives by any of the two step reaction sequences described above. Useful functionality to incorporate into the LTB4 antagonists includes the bromo, the nitro, the trialkylsilyl, the trialkyltin, and the boronic acid groups. The synthesis and application of each of these precursors is described in the references cited above.
The least complex means of radioiodination of the cyclic compounds of the present invention via isotopic labeling during the final stages of their preparation is the substitution of radioactive iodide for a stable iodine atom already present in the molecule. This can often be done by heating the compound with radioactive iodide in an appropriate solvent as described in Ellis et al., Aust. J. Chem., 26: 907 (1973). When applied to aromatic iodides, the extremely small quantities and low concentration of radioactive iodide employed leads to the incorporation of only modest specific activity.
The LTB4 antagonist compounds may also be isotopically iodo-labeled during the final stages of their preparation from the anilines by the Sandmeyer reaction as described in Ellis et al., Aust. J. Chem., 26: 907 (1973). This approach leads to a labeled cyclic compound with high specific activity. To avoid complications in the synthesis of the LTB4 antagonist compound, the nitro group provides an ideal synthon for the aniline.
Labeled iodo derivatives may also be readily prepared nonisotopically from the amino, hydroxy, or methoxy substituted cyclic compounds as described in Arora et al J. Med. Chem., 30:918 (1987). Electrophilic aromatic substitution reactions are enhanced by the presence of such electron-donating substituents.
Various procedures may also be employed in preparing the radiolabeled compounds of the invention where the radiolabel is a metal, such as where the radiolabel is technetium or indium. Exemplary procedures for such technetium or indium labeling are disclosed, for example, in Cerqueira et al., Circulation, Vol. 85, No. 1, pp. 298-304 (1992), Pak et al., J. Nucl. Med., Vol. 30, No. 5, p. 793, 36th Ann. Meet. Soc. Nucl. Med. (1989), Epps et al., J. Nucl. Med., Vol. 30, No. 5, p. 794, 36th Ann. Meet. Soc. Nucl. Med. (1989), Pak et al., J. Nucl. Med., Vol. 30, No. 5, p. 794, 36th Ann. Meet. Soc. Nucl. Med. (1989), and Dean et al., J. Nucl. Med., Vol. 30, No. 5, p. 794, 36th Ann. Meet. Soc. Nucl. Med. (1989), the disclosures of each of which are hereby incorporated herein by reference, in their entirety.
Preferred reagents of the present invention are comprised of chelators or radionuclide bonding units which are diaminedithiols, monoamine-monoamidedithiols, triamide-monothiols, monoamine-diamide-monothiols, diaminedioximes, or hydrazines. The chelators are generally tetradentate with donor atoms selected from nitrogen, oxygen and sulfur. More preferred reagents are comprised of chelators having amine nitrogen and thiol sulfur donor atoms and hydrazine bonding units. The thiol sulfur atoms and the hydrazines may bear a protecting group which can be displaced either prior to using the reagent to synthesize a radiopharmaceutical or preferrably in situ during the synthesis of the radiopharmaceutical.
Exemplary thiol protecting groups include those listed in Greene and Wuts, xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d John Wiley and Sons, New York (1991), the disclosure of which is hereby incorporated by reference. Any thiol protecting group known in the art can be used. Examples of thiol protecting groups include, but are not limited to, the following: acetamidomethyl, benzamidomethyl, 1-ethoxyethyl, benzoyl, and triphenylmethyl.
Exemplary protecting groups for hydrazine bonding units are hydrazones which can be aldehyde or ketone hydrazones having substituents selected from hydrogen, alkyl, aryl and heterocycle. Particularly preferred hydrazones are described in co-pending U.S. Ser. No. 08/476,296 the disclosue of which is herein incorporated by reference in its entirety.
The hydrazine bonding unit when bound to a metal radionuclide is termed a hydrazido, or diazenido group and serves as the point of attachment of the radionuclide to the remainder of the radiopharmaceutical. A diazenido group can be either terminal (only one atom of the group is bound to the radionuclide) or chelating. In order to have a chelating diazenido group at least one other atom of the group must also be bound to the radionuclide. The atoms bound to the metal are termed donor atoms.
The transition metal radionuclide is selected from the group: technetium-99 m, rhenium-186 and rhenium-188. For diagnostic purposes Tc-99 m is the preferred isotope. Its 6 hour half-life and 140 keV gamma ray emission energy are almost ideal for gamma scintigraphy using equipment and procedures well established for those skilled in the art. The rhenium isotopes also have gamma ray emission energies that are compatible with gamma scintigraphy, however, they also emit high energy beta particles that are more damaging to living tissues. These beta particle emissions can be utilized for therapeutic purposes, for example, cancer radiotherapy.
The coordination sphere of the radionuclide includes all the ligands or groups bound to the radionuclide. For a transition metal radionuclide to be stable it typically has a coordination number (number of donor atoms) comprised of an integer greater than or equal to 4 and less than or equal to 8; that is there are 4 to 8 atoms bound to the metal and it is said to have a complete coordination sphere. The requisite coordination number for a stable radionuclide complex is determined by the identity of the radionuclide, its oxidation state, and the type of donor atoms. If the chelator or bonding unit does not provide all of the atoms necessary to stabilize the metal radionuclide by completing its coordination sphere, the coordination sphere is completed by donor atoms from other ligands, termed ancillary or co-ligands, which can also be either terminal or chelating.
A large number of ligands can serve as ancillary or co-ligands, the choice of which is determined by a variety of considerations such as the ease of synthesis of the radiopharmaceutical, the chemical and physical properties of the ancillary ligand, the rate of formation, the yield, and the number of isomeric forms of the resulting radiopharmaceuticals, the ability to administer said ancillary or co-ligand to a patient without adverse physiological consequences to said patient, and the compatibility of the ligand in a lyophilized kit formulation. The charge and lipophilicity of the ancillary ligand will effect the charge and lipophilicity of the radiopharmaceuticals. For example, the use of 4,5-dihydroxy-1,3-benzene disulfonate results in radiopharmaceuticals with an additional two anionic groups because the sulfonate groups will be anionic under physiological conditions. The use of N-alkyl substituted 3,4-hydroxypyridinones results in radiopharmaceuticals with varying degrees of lipophilicity depending on the size of the alkyl substituents.
Preferred radiopharmaceuticals of the present invention are comprised of a hydrazido or diazenido bonding unit and an ancillary ligand, AL1, or a bonding unit and two types of ancillary AL1 and AL2, or a tetradentate chelator comprised of two nitrogen and two sulfur atoms. Ancillary ligands AL1 are comprised of two or more hard donor atoms such as oxygen and amine nitrogen (sp3 hydribidized). The donor atoms occupy at least two of the sites in the coordination sphere of the radionuclide metal; the ancillary ligand AL1 serves as one of the three ligands in the ternary ligand system. Examples of ancillary ligands AL1 include but are not limited to dioxygen ligands and functionalized aminocarboxylates. A large number of such ligands are available from commercial sources.
Ancillary dioxygen ligands include ligands that coordinate to the metal ion through at least two oxygen donor atoms. Examples include but are not limited to: glucoheptonate, gluconate, 2-hydroxyisobutyrate, lactate, tartrate, mannitol, glucarate, maltol, Kojic acid, 2,2-bis(hydroxymethyl)propionic acid, 4,5-dihydroxy-1,3-benzene disulfonate, or substituted or unsubstituted 1,2 or 3,4 hydroxypyridinones. (The names for the ligands in these examples refer to either the protonated or non-protonated forms of the ligands.)
Functionalized aminocarboxylates include ligands that have a combination of amine nitrogen and oxygen donor atoms. Examples include but are not limited to: iminodiacetic acid, 2,3-diaminopropionic acid, nitrilotriacetic acid, N,Nxe2x80x2-ethylenediamine diacetic acid, N,N,Nxe2x80x2-ethylenediamine triacetic acid, hydroxyethylethylenediamine triacetic acid, and N,Nxe2x80x2-ethylenediamine bis-hydroxyphenylglycine. (The names for the ligands in these examples refer to either the protonated or non-protonated forms of the ligands.)
A series of functionalized aminocarboxylates are disclosed by Bridger et. al. in U.S. Pat. No. 5,350,837, herein incorporated by reference, that result in improved rates of formation of technetium labeled hydrazino modified proteins. We have determined that certain of these aminocarboxylates result in improved yields of the radiopharmaceuticals of the present invention. The preferred ancillary ligands AL1 functionalized aminocarboxylates that are derivatives of glycine; the most preferred is tricine (tris(hydroxymethyl)methylglycine).
The most preferred radiopharmaceuticals of the present invention are comprised of a hydrazido or diazenido bonding unit and two types of ancillary designated AL1 and AL2, or a diaminedithiol chelator. The second type of ancillary ligands AL2 are comprised of one or more soft donor atoms selected from the group: phosphine phosphorus, arsine arsenic, imine nitrogen (sp2 hydridized), sulfur (sp2 hydridized) and carbon (sp hybridized); atoms which have p-acid character. Ligands AL2 can be monodentate, bidentate or tridentate, the denticity is defined by the number of donor atoms in the ligand. One of the two donor atoms in a bidentate ligand and one of the three donor atoms in a tridentate ligand must be a soft donor atom. We have disclosed in co-pending U.S. Ser. No. 08/415,908, and U.S. Ser. Nos. 60/013360 and 08/646,886, the disclosures of which are herein incorporated by reference in their entirety, that radiopharmaceuticals comprised of one or more ancillary or co-ligands AL2 are more stable compared to radiopharmaceuticals that are not comprised of one or more ancillary ligands, AL2; that is, they have a minimal number of isomeric forms, the relative ratios of which do not change significantly with time, and that remain substantially intact upon dilution.
The ligands AL2 that are comprised of phosphine or arsine donor atoms are trisubstituted phosphines, trisubstituted arsines, tetrasubstituted diphosphines and tetrasubstituted diarsines. The ligands AL2 that are comprised of imine nitrogen are unsaturated or aromatic nitrogen-containing, 5 or 6-membered heterocycles. The ligands that are comprised of sulfur (sp2 hybridized) donor atoms are thiocarbonyls, comprised of the moiety Cxe2x95x90S. The ligands comprised of carbon (sp hybridized) donor atoms are isonitriles, comprised of the moiety CNR, where R is an organic radical. A large number of such ligands are available from commercial sources. Isonitriles can be synthesized as described in European Patent 0107734 and in U.S. Pat. No. 4,988,827, herein incorporated by reference.
Preferred ancillary ligands AL2 are trisubstituted phosphines and unsaturated or aromatic 5 or 6 membered heterocycles. The most preferred ancillary ligands AL2 are trisubstituted phosphines and unsaturated 5 membered heterocycles.
The ancillary ligands AL2 may be substituted with alkyl, aryl, alkoxy, heterocycle, aralkyl, alkaryl and arylalkaryl groups and may or may not bear functional groups comprised of heteroatoms such as oxygen, nitrogen, phosphorus or sulfur. Examples of such functional groups include but are not limited to: hydroxyl, carboxyl, carboxamide, nitro, ether, ketone, amino, ammonium, sulfonate, sulfonamide, phosphonate, and phosphonamide. The functional groups may be chosen to alter the lipophilicity and water solubility of the ligands which may affect the biological properties of the radiopharmaceuticals, such as altering the distribution into non-target tissues, cells or fluids, and the mechanism and rate of elimination from the body.
The radiopharmaceuticals of the present invention comprised of a hydrazido or diazenido bonding unit can be easily prepared by admixing a salt of a radionuclide, a reagent of the present invention, an ancillary ligand AL1, an ancillary ligand AL2, and a reducing agent, in an aqueous solution at temperatures from 0 to 100xc2x0 C. The radiopharmaceuticals of the present invention comprised of a tetradentate chelator having two nitrogen and two sulfur atoms can be easily prepared by admixing a salt of a radionuclide, a reagent of the present invention, and a reducing agent, in an aqueous solution at temperatures from 0 to 100xc2x0 C.
When the bonding unit in the reagent of the present invention is present as a hydrazone group, then it must first be converted to a hydrazine, which may or may not be protonated, prior to complexation with the metal radionuclide. The conversion of the hydrazone group to the hydrazine can occur either prior to reaction with the radionuclide, in which case the radionuclide and the ancillary or co-ligand or ligands are combined not with the reagent but with a hydrolyzed form of the reagent bearing the chelator or bonding unit, or in the presence of the radionuclide in which case the reagent itself is combined with the radionuclide and the ancillary or co-ligand or ligands. In the latter case, the pH of the reaction mixture must be neutral or acidic.
Alternatively, the radiopharmaceuticals of the present invention comprised of a hydrazido or diazenido bonding unit can be prepared by first admixing a salt of a radionuclide, an ancillary ligand AL1, and a reducing agent in an aqueous solution at temperatures from 0 to 100xc2x0 C. to form an intermediate radionuclide complex with the ancillary ligand AL1 then adding a reagent of the present invention and an ancillary ligand AL2 and reacting further at temperatures from 0 to 100xc2x0 C.
Alternatively, the radiopharmaceuticals of the present invention comprised of a hydrazido or diazenido bonding unit can be prepared by first admixing a salt of 100xc2x0 C. to form an intermediate radionuclide complex with the ancillary ligand AL1 then adding a reagent of the present invention and an ancillary ligand AL2 and reacting further at temperatures from 0 to 100xc2x0 C.
Alternatively, the radiopharmaceuticals of the present invention comprised of a hydrazido or diazenido bonding unit can be prepared by first admixing a salt of a radionuclide, an ancillary ligand AL1, a reagent of the present invention, and a reducing agent in an aqueous solution at temperatures from 0 to 100xc2x0 C. to form an intermediate radionuclide complex, and then adding an ancillary ligand AL2 and reacting further at temperatures from 0 to 100xc2x0 C.
The total time of preparation will vary depending on the identity of the radionuclide, the identities and amounts of the reactants and the procedure used for the preparation. The preparations may be complete, resulting in  greater than 80% yield of the radiopharmaceutical, in 1 minute or may require more time. If higher purity radiopharmaceuticals are needed or desired, the products can be purified by any of a number of techniques well known to those skilled in the art such as liquid chromatography, solid phase extraction, solvent extraction, dialysis or ultrafiltration.
The technetium and rhenium radionuclides are preferably in the chemical form of pertechnetate or perrhenate and a pharmaceutically acceptable cation. The pertechnetate salt form is preferably sodium pertechnetate such as obtained from commercial Tc-99m generators. The amount of pertechnetate used to prepare the radiopharmaceuticals of the present invention can range from 0.1 mCi to 1 Ci, or more preferably from 1 to 200 mCi.
The amounts of the ancillary ligands AL1 used can range from 0.1 mg to 1 g, or more preferrably from 1 mg to 100 mg. The exact amount for a particular radiopharmaceutical is a function of identity of the radiopharmaceuticals of the present invention to be prepared, the procedure used and the amounts and identities of the other reactants. Too large an amount of AL1 will result in the formation of by-products comprised of technetium labeled AL1 without a biologically active molecule or by-products comprised of technetium labeled biologically active molecules with the ancillary ligand AL1 but without the ancillary ligand AL2. Too small an amount of AL1 will result in other by-products such as technetium labeled biologically active molecules with the ancillary ligand AL2 but without the ancillary ligand AL1, or reduced hydrolyzed technetium, or technetium colloid.
The amounts of the ancillary ligands AL2 used can range from 0.001 mg to 1 g, or more preferrably from 0.01 mg to 10 mg. The exact amount for a particular radiopharmaceutical is a function of the identity of the radiopharmaceuticals of the present invention to be prepared, the procedure used and the amounts and identities of the other reactants. Too large an amount of AL2 will result in the formation of by-products comprised of technetium labeled AL2 without a biologically active molecule or by-products comprised of technetium labeled biologically active molecules with the ancillary ligand AL2 but without the ancillary ligand AL1. If the reagent bears one or more substituents that are comprised of a soft donor atom, as defined above, at least a ten-fold molar excess of the ancillary ligand AL2 to the reagent of formula 2 is required to prevent the substituent from interfering with the coordination of the ancillary ligand AL2 to the metal radionuclide.
Suitable reducing agents for the synthesis of the radiopharmaceuticals of the present invention include stannous salts, dithionite or bisulfite salts, borohydride salts, and formamidinesulfinic acid, wherein the salts are of any pharmaceutically acceptable form. The preferred reducing agent is a stannous salt. The amount of a reducing agent used can range from 0.001 mg to 10 mg, or more preferably from 0.005 mg to 1 mg.
The specific structure of a radiopharmaceutical of the present invention comprised of a hydrazido or diazenido bonding unit will depend on the identity of the reagent of the present invention used, the identity of any ancillary ligand AL1, the identity of any ancillary ligand AL2, and the identity of the radionuclide. Radiopharmaceuticals comprised of a hydrazido or diazenido bonding unit synthesized using concentrations of reagents of  less than 100 xcexcg/mL, will be comprised of one hydrazido or diazenido group. Those synthesized using  greater than 1 mg/mL concentrations will be comprised of two hydrazido or diazenido groups from two reagent molecules. For most applications, only a limited amount of the biologically active molecule can be injected and not result in undesired side-effects, such as chemical toxicity, interference with a biological process or an altered biodistibution of the radiopharmaceutical. Therefore, the radiopharmaceuticals which require higher concentrations of the reagents comprised in part of the biologically active molecule, will have to be diluted or purified after synthesis to avoid such side-effects.
The identities and amounts used of the ancillary ligands AL1 and AL2 will determine the values of the variables y and z. The values of y and z can independently be an integer from 1 to 2. In combination, the values of y and z will result in a technetium coordination sphere that is made up of at least five and no more than seven donor atoms. For monodentate ancillary ligands AL2, z can be an integer from 1 to 2; for bidentate or tridentate ancillary ligands AL2, z is 1. The preferred combination for monodentate ligands is y equal to 1 or 2 and z equal to 1. The preferred combination for bidentate or tridentate ligands is y equal to 1 and z equal to 1.
Another aspect of the present invention are diagnostic kits for the preparation of radiopharmaceuticals useful as imaging agents for the inflammation and infection. Diagnostic kits of the present invention comprise one or more vials containing the sterile, non-pyrogenic, formulation comprised of a predetermined amount of a reagent of the present invention, one or two ancillary and optionally other components such as reducing agents, transfer ligands, buffers, lyophilization aids, stabilization aids, solubilization aids and bacteriostats. The inclusion of one or more optional components in the formulation will frequently improve the ease of synthesis of the radiopharmaceutical by the practising end user, the ease of manufacturing the kit, the shelf-life of the kit, or the stability and shelf-life of the radiopharmaceutical. The one or more vials that contain all or part of the formulation can independently be in the form of a sterile solution or a lyophilized solid.
Buffers useful in the preparation of radiopharmaceuticals and in diagnostic kits useful for the preparation of said radiopharmaceuticals include but are not limited to phosphate, citrate, sulfosalicylate, and acetate. A more complete list can be found in the United States Pharmacopeia.
Lyophilization aids useful in the preparation of diagnostic kits useful for the preparation of radiopharmaceuticals include but are not limited to mannitol, lactose, sorbitol, dextran, Ficoll, and polyvinylpyrrolidine(PVP).
Stabilization aids useful in the preparation of radiopharmaceuticals and in diagnostic kits useful for the preparation of said radiopharmaceuticals include but are not limited to ascorbic acid, cysteine, monothioglycerol, sodium bisulfite, sodium metabisulfite, gentisic acid, and inositol.
Solubilization aids useful in the preparation of radiopharmaceuticals and in diagnostic kits useful for the preparation of said radiopharmaceuticals include but are not limited to ethanol, glycerin, polyethylene glycol, propylene glycol, polyoxyethylene sorbitan monooleate, sorbitan monoloeate, polysorbates, poly(oxyethylene)poly(oxypropylene)poly(oxyethylene) block copolymers (Pluronics) and lecithin. Preferred solubilizing aids are polyethylene glycol, and Pluronics.
Bacteriostats useful in the preparation of radiopharmaceuticals and in diagnostic kits useful for the preparation of said radiopharmaceuticals include but are not limited to benzyl alcohol, benzalkonium chloride, chlorbutanol, and methyl, propyl or butyl paraben.
A component in a diagnostic kit can also serve more than one function. A reducing agent can also serve as a stabilization aid, a buffer can also serve as a transfer ligand, a lyophilization aid can also serve as a transfer, ancillary or co-ligand and so forth.
The predetermined amounts of each component in the formulation are determined by a variety of considerations that are in some cases specific for that component and in other cases dependent on the amount of another component or the presence and amount of an optional component. In general, the minimal amount of each component is used that will give the desired effect of the formulation. The desired effect of the formulation is that the practising end user can synthesize the radiopharmaceutical and have a high degree of certainty that the radiopharmaceutical can be safely injected into a patient and will provide diagnostic information about the disease state of that patient.
Another aspect of the present invention contemplates a method of imaging the site of infection or inflammation in a patient involving: (1) synthesizing a radiopharmaceutical using a reagent of the present invention capable of localizing at sites of infection or inflammation; (2) administering said radiopharmaceutical to a patient by injection or infusion; (3) imaging the patient using either planar or SPECT gamma scintigraphy.
A further aim of the present invention is the use of LTB4-targeted radiopharmaceuticals in a dual isotope imaging procedure in conjunction with a perfusion imaging radiopharmaceutical. In this dual isotope procedure a scintigraphic image of the radiolabeled LTB4 receptor-binding compound is acquired at the same time as a scintigraphic image of a radiolabeled cardiac perfusion imaging agent. This simultaneous dual isotope imaging is done by utilizing radioisotopes bound to the LTB4 antagonist and the perfusion imaging agent which have spectrally separable gamma emission energies. For example, a Tc99m cardiac perfusion imaging agent (such as Tc99 m-Sestamibi) or T1201 (as Thallous Chloride), and an In111-labeled LTB4 antagonist compound are imaged simultaneously with a standard gamma camera. This is possible because the Tc99m gamma energy of xcx9c140 KeV or the T1201 gamma energy of xcx9c80 KeV are easily separable from the In111 gamma energies of xcx9c160 KeV and 250 KeV. This simultaneous imaging of cardiac perfusion and cardiac inflammation (as evidenced by the LTB4 compound localization) is extremely useful for improved anatomic assessment of the location of LTB4 receptor distribution in the heart based on the comparison to the perfusion distribution seen on the Tc99 m-Sestamibi or T1201 image. It also allows for the evaluation of a match or mismatch between cardiac perfusion and the cardiac inflammation image. The simultaneous imaging of perfusion and inflammation allows a more complete assessment of the underlying cardiac disease, both in terms of blood flow alterations and inflammatory changes, in a single imaging session on a patient.
The simultaneous dual-isotope imaging of perfusion and inflammation allows for the localization of sites of ischemic tissue injury (e.g. reperfusion injury), vulnerable plaque, bacterial endocarditis or cardiac transplant rejection to be correlated with cardiac perfusion during one imaging session. In addition, the imaging of inflammatory response associated with ischemic tissue damage (such as in reperfusion injury) and myocardial blood flow (from the perfusion imaging agent) is extremely useful in characterizing the severity and extent/location of inflammation. This simultaneous imaging procedure is also useful in monitoring responses to therapy for reducing cardiac inflammation simultaneously with monitoring changes in myocardial blood flow.
The simultaneous imaging of different radioisotopes/radiopharmaceuticals for localization of inflammation and cardiac perfusion allows a more exact registration of the images than would be possible when comparing two serially-acquired images. It is also a more efficient use of imaging equipment and health care personnel time and resources. This simultaneous imaging will save several hundred dollars per study relative to two separate imaging procedures.
The simultaneous imaging of different radioisotopically-labeled radiopharmaceuticals for organ perfusion and a biospecific target in patients is not new. For example, Antunes, et al., have demonstrated that it is possible to image myocardial infarction with an In111-antimyosin antibody along with the imaging of cardiac perfusion with T1201. However, the dual isotope imaging of the present invention is new, because it is the first reported approach to the simultaneous, dual isotope imaging of a radiolabeled inflammation imaging compound and a perfusion imaging compound. The combination of LTB4 antagonist scintigraphic imaging with perfusion imaging provides the imaging physician with an extraordinary amount of clinical information regarding, for example, reperfusion injury to transiently ischemic tissue or inflammation associated with active atherosclerotic plaque and organ blood flow changes in a single imaging session.
The radiopharmaceuticals are administered by intravenous injection, usually in saline solution, at a dose of 1 to 100 mCi per 70 kg body weight, or preferably at a dose of 5 to 50 mCi. Imaging is performed using known procedures.